EP1019430B1 - Method for isolating a nucleic acid - Google Patents

Method for isolating a nucleic acid Download PDF

Info

Publication number
EP1019430B1
EP1019430B1 EP98952670A EP98952670A EP1019430B1 EP 1019430 B1 EP1019430 B1 EP 1019430B1 EP 98952670 A EP98952670 A EP 98952670A EP 98952670 A EP98952670 A EP 98952670A EP 1019430 B1 EP1019430 B1 EP 1019430B1
Authority
EP
European Patent Office
Prior art keywords
process according
sample
glass particles
analyte
carried out
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Lifetime
Application number
EP98952670A
Other languages
German (de)
French (fr)
Other versions
EP1019430A2 (en
Inventor
Jörg KLEIBER
Christine Markert-Hahn
Herbert Harttig
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Roche Diagnostics GmbH
Original Assignee
Roche Diagnostics GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Roche Diagnostics GmbH filed Critical Roche Diagnostics GmbH
Priority to EP07003878A priority Critical patent/EP1783135B1/en
Publication of EP1019430A2 publication Critical patent/EP1019430A2/en
Application granted granted Critical
Publication of EP1019430B1 publication Critical patent/EP1019430B1/en
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

Links

Images

Classifications

    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01F—MAGNETS; INDUCTANCES; TRANSFORMERS; SELECTION OF MATERIALS FOR THEIR MAGNETIC PROPERTIES
    • H01F1/00—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties
    • H01F1/01—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties of inorganic materials
    • H01F1/03—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties of inorganic materials characterised by their coercivity
    • H01F1/032—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties of inorganic materials characterised by their coercivity of hard-magnetic materials
    • H01F1/10—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties of inorganic materials characterised by their coercivity of hard-magnetic materials non-metallic substances, e.g. ferrites, e.g. [(Ba,Sr)O(Fe2O3)6] ferrites with hexagonal structure
    • H01F1/11—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties of inorganic materials characterised by their coercivity of hard-magnetic materials non-metallic substances, e.g. ferrites, e.g. [(Ba,Sr)O(Fe2O3)6] ferrites with hexagonal structure in the form of particles
    • H01F1/112—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties of inorganic materials characterised by their coercivity of hard-magnetic materials non-metallic substances, e.g. ferrites, e.g. [(Ba,Sr)O(Fe2O3)6] ferrites with hexagonal structure in the form of particles with a skin
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C1/00—Magnetic separation
    • B03C1/005—Pretreatment specially adapted for magnetic separation
    • B03C1/01—Pretreatment specially adapted for magnetic separation by addition of magnetic adjuvants
    • C—CHEMISTRY; METALLURGY
    • C03—GLASS; MINERAL OR SLAG WOOL
    • C03C—CHEMICAL COMPOSITION OF GLASSES, GLAZES OR VITREOUS ENAMELS; SURFACE TREATMENT OF GLASS; SURFACE TREATMENT OF FIBRES OR FILAMENTS MADE FROM GLASS, MINERALS OR SLAGS; JOINING GLASS TO GLASS OR OTHER MATERIALS
    • C03C14/00—Glass compositions containing a non-glass component, e.g. compositions containing fibres, filaments, whiskers, platelets, or the like, dispersed in a glass matrix
    • C03C14/004—Glass compositions containing a non-glass component, e.g. compositions containing fibres, filaments, whiskers, platelets, or the like, dispersed in a glass matrix the non-glass component being in the form of particles or flakes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C07H1/06—Separation; Purification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C07H1/06—Separation; Purification
    • C07H1/08—Separation; Purification from natural products
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/0099—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor comprising robots or similar manipulators
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/0098—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor involving analyte bound to insoluble magnetic carrier, e.g. using magnetic separation

Definitions

  • the invention relates to a method for preparing biological samples for the subsequent detection of an analyte, in particular a nucleic acid, in this sample. Furthermore, reagent kits are provided.
  • analyte In a method for detecting an analyte in a biological sample, special requirements are often placed on sample preparation. On the one hand, the analyte is often present in very low concentrations and, on the other hand, there are often many other substances in the sample which can impair the isolation or determination of the analyte.
  • WO 36/41811 discloses a method for isolating an analyte, in particular a nucleic acid, from a biological sample, wherein the sample containing the analyte in a liquid comprises magnetic particles having an outer glass surface that is substantially nonporous or pores of a diameter of ⁇ 10 nm, under conditions in which the analyte binds to the particle surface, and the bound analyte is separated from the sample liquid.
  • the magnetic particles are used here in solid form and not in the form of a suspension.
  • the glass surface consists of a borosilicate glass. This in WO 96/46811 described method is very suitable for the purification of an analyte from a biological sample. However, it is not readily usable for automated sample preparation.
  • the object underlying the present invention was therefore to provide a new sample preparation method in which the disadvantages of the prior art are at least partially eliminated.
  • the new method should be automated and have the simplest possible temperature profile.
  • the method of the invention is based on the selective binding of analytes to a solid adsorption matrix in the presence of a sample digestion buffer, wherein the analyte, which is preferably a nucleic acid such as DNA, e.g. chromosomal DNA, fragmented chromosomal DNA, plasmid DNA, viral DNA, etc., or RNA, e.g. mRNA, tRNA, rRNA or viral. RNA, etc., is separated from contaminants of the sample, such as proteins or cell debris.
  • the sample may be any biological sample, e.g. a body fluid such as blood, plasma, urine etc, a tissue sample, a sample of cultured cells or the like.
  • the adsorption matrix used in the method according to the invention is capable of ensuring a binding of the analyte which is largely selective under the reaction conditions.
  • magnetic glass particles are used, in particular those in WO 96/41811 described magnetic particles with an outer Glass surface which is substantially free of pores or has pores of a diameter of less than 10 nm.
  • Particularly preferred are ferromagnetic particles having a particle size between 10 and 60 microns.
  • Such particles may, for example, contain a core of mica and magnetic particles immobilized thereon, which is enclosed by the glass layer.
  • the magnetic particles are presented in solid form, for example as tablets or powders, in the respective reaction vessels used, the magnetic particles are used according to the invention in the form of an alcoholic suspension.
  • Particularly suitable are alcoholic suspensions with a concentration of about 5 to 20 mg / ml proved.
  • glass particles whose glass phase comprises the following metal oxides.
  • SiO 2 , B 2 O 3 alkali metal oxide, for example K 2 O or / and Na 2 O and optionally Al 2 O 3 and an alkaline earth metal oxide, for example CaO.
  • the proportions of these metal oxides are preferably as follows: 50 to 95 mol% SiO 2 , 0.2 to 30 mol% B 2 O 3 , 0 to 10 mol% Al 2 O 3 , 0 to 20 mol% alkaline earth metal oxide and 0.2 to 20 mol% alkali metal oxide, wherein the percentages are based on the total weight of the glass phase.
  • RNA for example, a glass phase containing SiO 2 , B 2 O 3 , K 2 O, Al 2 O 3 and CaO has proven to be particularly suitable.
  • the adsorption matrix is preferably added in an amount which corresponds to the minimum amount required for quantitatively binding the analyte present in the sample, in particular a nucleic acid, or slightly larger, preferably not more than 50% and particularly preferably not more than 20% this amount is.
  • the expected amount of nucleic acid in different types of samples, unless already known, may be determined in advance by conventional techniques, e.g. Phenol / chloroform extraction and subsequent measurement of the optical density can be determined.
  • Step (a) of the process of the invention comprises digesting the sample in a reaction vessel. This digestion is generally carried out by lysing the cells present in the sample under denaturing conditions, e.g. by adding a protease and a denaturation buffer.
  • the proteinase used is preferably proteinase K, pronase, elastase or / and lysozyme. Particularly preferred is the use of proteinase K.
  • Protease digestion is carried out in a denaturation buffer containing a chaotropic compound, e.g. Urea or urea derivatives, preferably a chaotropic salt, more preferably a guanidinium salt such as guanidinium hydrochloride (in particular for the isolation of DNA) or guanidinium thiocyanate (in particular for the isolation of RNA) or a perchlorate or iodide.
  • a chaotropic compound e.g. Urea or urea derivatives
  • a chaotropic salt more preferably a guanidinium salt such as guanidinium hydrochloride (in particular for the isolation of DNA) or guanidinium thiocyanate (in particular for the isolation of RNA) or a perchlorate or iodide.
  • concentrations in the range of 1 to 3 mol / l are preferred.
  • step (c) the selective binding of the analyte to the adsorption matrix is carried out by incubation in the digestion buffer, preferably under chaotropic conditions.
  • Step (d) of the method of the invention comprises separating unbound sample components from the adsorption matrix.
  • the unbound sample components are removed from the reactor vessel. This can be done by optionally adding and removing a washing buffer, preferably containing at least 50% (v / v) and more preferably at least 60% (v / v) of a water-miscible organic solvent such as ethanol, propanol and acetone contains.
  • a washing buffer preferably containing at least 50% (v / v) and more preferably at least 60% (v / v) of a water-miscible organic solvent such as ethanol, propanol and acetone contains.
  • the steps (c), (d) or / and (e) of the process according to the invention are preferably carried out with continuous or interval-like mixing (ie phases during which mixing takes place with phases in which the reaction vessel is at rest) without Addition of external funds.
  • This mixing preferably takes place by rotation of the reaction vessel about its longitudinal axis with repeated reversal of the direction of rotation.
  • the mixing vessel is rotated exactly about its longitudinal axis and the change of direction is performed so that the Meniskusauslenkung the liquid remains below a predetermined separation number.
  • Such mixing methods are in WO91 / 15768 and EP-A-0 435 481 described.
  • the duration for steps (c) or / and (e) is preferably at most 20 minutes and comprises continuous mixing or interval mixing in short cycles, preferably in short cycles of preferably maximally 2 minutes. Particularly good results were obtained by interval mixing in a one-minute cycle comprising 20 sec of mixing and 40 sec of rest.
  • the addition of liquids into the reaction vessel or the aspiration of liquids from it can be carried out with continuous mixing, wherein the particles are held in the reaction vessel during the suction by switching on the magnet.
  • the method according to the invention can be flexibly adjusted to different types of samples.
  • a constant distribution of the magnetic particles in the liquid phase is constantly ensured.
  • Step (e) of the method according to the invention comprises the elution of the analyte from the adsorption matrix.
  • a low-salt buffer substantially free of organic solvents can be used.
  • the elution buffer may contain additional reagents, such as enzymes, e.g. enzymes used to manipulate nucleic acids, such as RNases, DNases, restriction endonucleases, ligases, terminal transferases and / or polymerases.
  • enzymes e.g. enzymes used to manipulate nucleic acids, such as RNases, DNases, restriction endonucleases, ligases, terminal transferases and / or polymerases.
  • the analyte is a DNA
  • DNase-free RNase may be added during elution to reduce the level of unwanted RNA.
  • analyte is an RNA
  • an RNase-free DNase can be added during elution.
  • other enzymes such as restriction endonucleases, etc. may also be added.
  • a nucleic acid amplification master mix containing the amplification buffer, nucleotides, primers, polymerase and buffer salts may also be added during the elution.
  • Step (f) of the process of the invention comprises separating the eluate from the adsorption matrix.
  • This separation can be carried out in the usual way, for example by sedimentation, but preferably by magnetic separation.
  • the analytes isolated by the method according to the invention can then be further processed in a known manner, in the case of nucleic acids, e.g. by amplification and subsequent detection, detection without prior amplification or sequencing.
  • aliquots of the eluate itself may be supplied to determinations of different analytes, e.g. various viruses, such as HIV, HCV and HBV.
  • An important feature of the process of the invention is that many, or even all, of the steps are conducted at substantially the same temperature, i. within a temperature range of ⁇ 2.5 ° C.
  • this temperature is in the range of room temperature to 70 ° C, more preferably from room temperature to 40 ° C, most preferably at room temperature, i. about 18 to 32 ° C.
  • at least the steps (c) of the adsorption and (d) of the washing are carried out at this temperature.
  • Particular preference is also given to carrying out other steps, in particular steps (a) of digesting and / or (e) elution at this temperature.
  • the entire sample preparation may be done at a uniform temperature.
  • an additional post-treatment step may be carried out at elevated temperature, whereby the yields in the case of certain analytes are improved during an amplification.
  • Other analytes may require pretreatment and / or elution at elevated temperature.
  • the elevated temperature is preferably in the range of more than 40 ° C to 95 ° C, z.b. about 70 ° C.
  • the implementation of the method according to the invention is preferably carried out in an automated device. Examples of such devices are described below. Furthermore, it is preferred that the inventive A method for sample preparation, at least steps (a) to (e) are carried out in a single reaction vessel, ie that no transfer to another reaction vessel takes place. This results in a considerable simplification of the method and also leads to a reduced risk of contamination.
  • the apparatus is preferably designed so that a single reaction vessel for carrying out the four main steps of sample preparation, namely digestion of a sample or lysis, adsorption of the released analyte, e.g. a nucleic acid, to a solid adsorption matrix, e.g. magnetic glass particles, washing the adsorption matrix and elution of the analyte is provided by the adsorption matrix.
  • sample preparation namely digestion of a sample or lysis, adsorption of the released analyte, e.g. a nucleic acid, to a solid adsorption matrix, e.g. magnetic glass particles, washing the adsorption matrix and elution of the analyte is provided by the adsorption matrix.
  • the device is designed so that the first receiving device for receiving the reaction vessels for sample preparation is provided at least for the adsorption of the analyte to the solid adsorption matrix and for washing the adsorption matrix.
  • the first receiving device is further provided for digesting the sample and / or for eluting the analyte from the adsorption matrix.
  • the reaction vessels for sample preparation have a volume of preferably at least 1 ml, e.g. 1-5 ml.
  • the second receptacle is intended for reaction vessels for storage or / and further processing of the analyte, e.g. PCR vessels, which usually have a different shape from the reaction vessels used for sample preparation.
  • the reaction vessels for storage and / or further processing have a volume of preferably up to 500 ⁇ l, e.g. 50-200 ⁇ l.
  • the second receptacle may contain vessels for reagents needed for further processing of the sample containing the analyte, e.g. a PCR master mix.
  • the apparatus may be configured such that one or more steps of sample preparation and / or an aftertreatment step may be performed at an elevated temperature in the second receiving means.
  • the second receiving device may be provided for receiving reaction vessels for at least one treatment step, which is selected from the digestion of the sample, the Elution of the sample from the adsorption matrix and post-treatment step after elution.
  • the first receiving device preferably comprises means for magnetic separation. Furthermore, it is preferred that the first receiving means comprises means for mixing the reaction vessels, in particular by rotating about their longitudinal axis. If appropriate, such means can also be provided for the second receiving device.
  • the device generally comprises automatic pipetting devices and optionally means for transporting reaction vessels, e.g. B. between first and second receiving device.
  • a lid opening and closing unit can be integrated.
  • the device (1) contains a receiving device for reagents (2), a receptacle for reaction preparation tubes (3) with the functions of mixing and magnetic separation, preferably for a temperature of ⁇ 40 ° C. and more preferably Room temperature is provided. Furthermore, the device contains a receiving station for further reaction vessels (4a), for example for PCR vessels, which has a temperature of 4 ° C to room temperature. Furthermore, the device contains automated devices for the pipetting and handling of reaction vessels (5), which allow movements in the X, Y and Z directions.
  • the four main steps of sample preparation take place in the first receiving device in a single reaction vessel.
  • the vessels are transferred to a respective device, eg a thermal cycler (not shown).
  • the device contains a second receiving device (4b), which is intended to receive further processing reaction vessels, such as PCR vessels, and setting a temperature of 4 ° C (cooling of the PCR master mix) to 95 ° C. for heating the eluate after elution from the adsorption matrix.
  • a lid heater is preferred.
  • a second receptacle for reaction vessels (4c) is provided, which is provided for receiving PCR vessels and sample preparation vessels.
  • cooling e.g. to 4 ° C
  • heating e.g. to 95 ° C
  • a lid heater is preferred to avoid the formation of condensation on the lid of reaction vessels.
  • the first receiving means is provided for setting a temperature in the range of ⁇ 70 ° C.
  • the second receiver as shown in Figure 3, is suitable for cooling and heating sample processing and sample preparation vessels.
  • the sols were then subjected to a spray-drying process.
  • the powder obtained by the desiccation was subjected to fine particle separation by sedimentation, temperature treatment under nitrogen atmosphere (60 l / h volume flow) at a heating rate of 1 K / min, and held for 1 hour at a compression temperature in the range of 600 to 700 ° C for one hour. Subsequently, the furnace was cooled to 300 ° C and rinsed at this temperature for 1 h with oxygen. After cooling to room temperature, the magnetic glass particles were removed and placed on a 50 micron sieve and separated to separate the coarse fraction.
  • the magnetic glass particles obtained from sol 1 are particularly suitable for the isolation of DNA.
  • the glass particles obtained from sol 2 are particularly suitable for the isolation of RNA.
  • nucleic acids obtained in this way can be used immediately after elution for an amplification by PCR, a restriction cleavage or a Southern blot can be used.
  • the kit components are stable and can be stored at room temperature. After dissolution of proteinase K in water, the solution should be aliquoted and stored at -20 ° C. The frozen solution is stable for 12 months.
  • the above protocol may also be used in accordance with microtiter plates, eg Wohllochmikrotiterplatten (eg Ritter, HJ Bioanalytic).
  • the MGPs are concentrated by transferring the sample to a magnetic separator. After one minute, the supernatant is completely pipetted off.
  • 0.5 ml wash buffer is pipetted to the MGPs.
  • the sample is vortexed and then transferred to the magnetic separator.
  • the supernatant is pipetted off after 1 min.
  • the washing procedure is repeated twice more.
  • elution buffer 200 ml of elution buffer are added to the MGP.
  • the sample is incubated for 10 min at 70 ° C on a thermo-mixer at 1400 RPM. Condensation is collected by short centrifugation. The sample is transferred to the magnetic separator and after 1 min 180 ul eluate removed. The eluate is pipetted into a new reaction vessel and stored at 4 ° C (for a storage period ⁇ 24 h) or at -20 ° C (for a longer storage period).
  • PCR For the PCR, 50 ⁇ l of eluate are used. The evaluation is carried out by electrochemiluminescence.
  • FIG. 4 shows a comparison of the determination of chlamydia (sample: 100 elemental antibodies per 100 ml urine, sixfold determination) between the standard manual protocol (vortex) and the semi-automated method (MTM). It can be seen that the automation does not affect the sensitivity.
  • Sample preparation is carried out as described under point 3.2. However, lysis and elution are carried out at room temperature.
  • Sample preparation is carried out as described under point 3.3. After elution, incubate for 10 min at 70 ° C.
  • FIG. 5 shows a comparison of the chlamydia determination (samples: SWE1, O Chlamydia Elemental Antibody (EAC) per mL urine, SWE 2: 10 EAK, SWE 3: 100 EAK and SWE 4: 1000 EAC each per mL urine) between those in the points 3.2, 3.3 and 3.4 described sample preparation protocols.
  • EAC Elemental Antibody
  • the sample preparation process can be significantly simplified, because the steps of lysis, adsorption, washing and elution can be carried out at temperatures ⁇ 40 ° c, which simplifies automation, since no Deckelallestraung and temperature regulation is required.
  • Frozen plasma is thawed for 5 min at 37 ° C and cooled on ice for further processing.
  • a proteinase K solution 25 mg / ml
  • 50 ⁇ l of a proteinase K solution 25 mg / ml
  • 250 .mu.l sample are added and vortexed.
  • 300 ⁇ l of lysis buffer are added and vortexed again.
  • washing buffer To the MGP is added 750 ⁇ l washing buffer.
  • the MGPs are resuspended and separated as previously described. The washing procedure is repeated four times, at the end of which the washing buffer is carefully removed.
  • the sample preparation is carried out as described in section 4.1, except that the mixing and the temperature control took place on a mixing and tempering module.
  • the sample preparation is essentially as described in section 4.2, except that all steps are carried out at room temperature.
  • the incubation period for lysis, adsorption and elution is 15 min.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • Analytical Chemistry (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Plant Pathology (AREA)
  • Immunology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Dermatology (AREA)
  • Power Engineering (AREA)
  • Ceramic Engineering (AREA)
  • General Chemical & Material Sciences (AREA)
  • Geochemistry & Mineralogy (AREA)
  • Materials Engineering (AREA)
  • Dispersion Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Robotics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Sampling And Sample Adjustment (AREA)
  • Saccharide Compounds (AREA)
  • Glass Compositions (AREA)
  • Hard Magnetic Materials (AREA)

Abstract

Isolation of an analyte (I) from a biological sample comprising: (i) decomposing the sample in a reaction vessel; (ii) adding a solid adsorption matrix (A); (iii) incubating so that (I) binds to (A); (iv) removing non-bound components from the vessel; (v) incubating so that (I) is eluted from (A); and (vi) separating the eluate from (A). Independent claims are also included for the following: (1) reagent kits for the process comprising protease; buffers for sample decomposition, washing and elution, and a suspension of magnetic glass particles; (2) apparatus for the process; and (3) magnetic glass particles (B) comprising a magnetic core and glass shell containing silica, boron oxide and alkali metal oxide, optionally also alumina and alkaline earth metal oxide.

Description

Die Erfindung betrifft ein Verfahren zur Vorbereitung biologischer Proben für den anschließenden Nachweis eines Analyten, insbesondere einer Nukleinsäure, in dieser Probe. Weiterhin werden Reagenzienkits bereitgestellt.The invention relates to a method for preparing biological samples for the subsequent detection of an analyte, in particular a nucleic acid, in this sample. Furthermore, reagent kits are provided.

Bei einem Verfahren zum Nachweis eines Analyten in einer biologischen Probe werden an die Probenvorbereitung oft besondere Anforderungen gestellt. Zum einen ist der Analyt oft in sehr geringer Konzentration vorhanden und zum anderen finden sich oft viele andere Substanzen in der Probe, die die Isolierung bzw. Bestimmung des Analyten beeinträchtigen können.In a method for detecting an analyte in a biological sample, special requirements are often placed on sample preparation. On the one hand, the analyte is often present in very low concentrations and, on the other hand, there are often many other substances in the sample which can impair the isolation or determination of the analyte.

WO 36/41811 offenbart ein Verfahren zur Isolierung eines Analyten, insbesondere einer Nukleinsäure, aus einer biologischen Probe, wobei die Probe, die den Analyten in einer Flüssigkeit enthält, mit magnetischen Partikeln, die eine äußere Glasoberfläche, die im wesentlichen porenfrei ist oder Poren eines Durchmessers von < 10 nm aufweist, in Kontakt gebracht wird, unter Bedingungen, bei denen der Analyt an die Partikeloberfläche bindet, und der gebundene Analyt von der Probenflüssigkeit abgetrennt wird. Die magnetischen Partikel werden hier in fester Form und nicht in Form einer Suspension eingesetzt. Die Glasoberfläche besteht aus einem Borosilikatglas. Das in WO 96/46811 beschriebene Verfahren eignet sich sehr gut zur Aufreinigung eines Analyten aus einer biologischen Probe. Es ist jedoch nicht ohne weiteres für eine automatisierte Probenvorbereitung einsetzbar. WO 36/41811 discloses a method for isolating an analyte, in particular a nucleic acid, from a biological sample, wherein the sample containing the analyte in a liquid comprises magnetic particles having an outer glass surface that is substantially nonporous or pores of a diameter of <10 nm, under conditions in which the analyte binds to the particle surface, and the bound analyte is separated from the sample liquid. The magnetic particles are used here in solid form and not in the form of a suspension. The glass surface consists of a borosilicate glass. This in WO 96/46811 described method is very suitable for the purification of an analyte from a biological sample. However, it is not readily usable for automated sample preparation.

Boom et al. (J. Clin. Microbiol. 28 (1990), 495-503 ) beschreiben ebenfalls ein Protokoll für Aufreinigung von Nukleinsäuren aus einer biologischen Probe unter Verwendung größenfraktionierter Siliciumoxidteilchen. Dieses Verfahren ist jedoch umständlich, nicht für eine Automatisierung geeignet und enthält darüber hinaus die Gefahr von Verschleppungen. Boom et al. (J. Clin. Microbiol., 28, 495-503, 1990) ) also describe a protocol for purification of nucleic acids from a biological sample using size fractionated silica particles. This However, the process is cumbersome, not suitable for automation and also contains the risk of carryover.

Bei einer in EP-A-0 757 106 beschriebenen Methode zur Extraktion von Nukleinsäuren wird eine Probe lysiert, die in der Probe vorhandenen Nukleinsäuren an superparamagnetische Metallteilchen gebunden, diese mit einer Pipette aus dem Probengefäß entfernt und somit von den übrigen Probenbestandteilen abgetrennt. Dieses Verfahren hat den Nachteil, daß aufgrund der Notwendigkeit, den Analyten mit einer Pipette aus der Probe zu entfernen, Verluste auftreten können. Darüber hinaus beinhaltet die Verwendung mehrerer Reaktionsgefäße die Gefahr von Verschleppungen und Kontaminationen.At an in EP-A-0 757 106 described method for the extraction of nucleic acids, a sample is lysed, bound the nucleic acids present in the sample of superparamagnetic metal particles, these removed with a pipette from the sample vessel and thus separated from the other sample components. This method has the disadvantage that losses may occur due to the need to remove the analyte from the sample with a pipette. In addition, the use of multiple reaction vessels involves the risk of carryover and contamination.

Die der vorliegenden Erfindung zugrundeliegende Aufgabe bestand somit darin, ein neues Probenvorbereitungsverfahren bereitzustellen, bei dem die Nachteile des Standes der Technik mindestens teilweise beseitigt sind. Insbesondere soll das neue Verfahren automatisierbar sein und ein möglichst einfaches Temperaturprofil aufweisen.The object underlying the present invention was therefore to provide a new sample preparation method in which the disadvantages of the prior art are at least partially eliminated. In particular, the new method should be automated and have the simplest possible temperature profile.

Diese Aufgabe wird gelöst durch Verfahren zur Isolierung eines Analyten aus einer biologischen Probe, umfassend die Schritte:

  1. (a) Aufschließen der Probe in einem Reaktionsgefäß,
  2. (b) Zugeben von magnetischen Glaspartikel in Form einer alkoholischen Suspension.
  3. (c) Inkubieren unter Bedingungen, bei denen der Analyt an die Glaspartikel bindet,
  4. (d) Entfernen nichtgebundener Probenbestandteile aus dem Reaktionsgefäß,
  5. (e) Inkubieren unter Bedingungen, bei denen der Analyt von den Glaspartikeln eluiert wird, und
  6. (f) Abtrennen des Eluats von den Glaspartikeln,
wobei viele oder alle Schritte bei im wesentlichen der gleichen Temperatur durchgeführt werden, die vorzugsweise im Bereich von Raumtemperatur bis 40°C liegt.This object is achieved by methods for isolating an analyte from a biological sample, comprising the steps:
  1. (a) digesting the sample in a reaction vessel,
  2. (b) adding magnetic glass particles in the form of an alcoholic suspension.
  3. (c) incubating under conditions in which the analyte binds to the glass particles,
  4. (d) removing unbound sample components from the reaction vessel,
  5. (e) incubating under conditions in which the analyte is eluted from the glass particles, and
  6. (f) separating the eluate from the glass particles,
wherein many or all of the steps are carried out at substantially the same temperature, preferably ranging from room temperature to 40 ° C.

Das erfindungsgemäße Verfahren beruht auf der selektiven Bindung von Analyten an eine feste Adsorptionsmatrix in Gegenwart eines Probenaufschlußpuffers, wobei der Analyt, bei dem es sich vorzugsweise um eine Nukleinsäure wie DNA, z.B. chromosomale DNA, fragmentierte chromosomale DNA, Plasmid-DNA, virale DNA etc., oder RNA, z.B. mRNA, tRNA, rRNA oder virale. RNA etc. handelt, von Verunreinigungen der Probe wie etwa Proteinen oder Zelltrümmern abgetrennt wird. Die Probe kann eine beliebige biologische Probe sein, z.B. eine Körperflüssigkeit wie Blut, Plasma, Urin etc, eine Gewebeprobe, eine Probe von kultivierten Zellen oder ähnliches.The method of the invention is based on the selective binding of analytes to a solid adsorption matrix in the presence of a sample digestion buffer, wherein the analyte, which is preferably a nucleic acid such as DNA, e.g. chromosomal DNA, fragmented chromosomal DNA, plasmid DNA, viral DNA, etc., or RNA, e.g. mRNA, tRNA, rRNA or viral. RNA, etc., is separated from contaminants of the sample, such as proteins or cell debris. The sample may be any biological sample, e.g. a body fluid such as blood, plasma, urine etc, a tissue sample, a sample of cultured cells or the like.

Die beim erfindungsgemäßen Verfahren verwendete Adsorptionsmatrix ist in der Lage, eine unter den Reaktionsbedingungen weitgehend selektive Bindung des Analyten zu gewährleisten. Erfindungsgemäß verwendet man magnetische Glaspartikel, insbesondere die in WO 96/41811 beschriebenen magnetischen Partikel mit einer äußeren Glasoberfläche, die im wesentlichen porenfrei ist oder Poren eines Durchmessers von weniger als 10 nm aufweist. Besonders bevorzugt sind ferromagnetische Partikel, die eine Korngröße zwischen 10 und 60 µm aufweisen. Solche Partikel können beispielsweise einen Kern aus Glimmer und darauf immobilisierten Magnetpartikeln enthalten, der von der Glasschicht umschlossen ist. Während in WO 96/4181 die magnetischen Partikel in fester Form, z.B. als Tabletten oder Pulver, in den jeweils verwendeten Reaktionsgefäßen vorgelegt werden, setzt man die magnetischen Partikeln erfindungsgemäß in Form einer alkoholischer Suspension ein. B sonders geeignet haben sich alkoholische Suspensionen mit einer Konzentration von etwa 5 bis 20 mg/ml erwiesen. Überraschenderweise wurde festgestellt, daß trotz der hohen spezifischen Dichte der magnetischen Glaspartikel die Suspension mit hoher Reproduzierbarkeit aus einem Vorratsbehälter abgezogen werden kann, wodurch eine Automatisierbarkeit der Verfahrensführung ermöglicht wird.The adsorption matrix used in the method according to the invention is capable of ensuring a binding of the analyte which is largely selective under the reaction conditions. According to the invention, magnetic glass particles are used, in particular those in WO 96/41811 described magnetic particles with an outer Glass surface which is substantially free of pores or has pores of a diameter of less than 10 nm. Particularly preferred are ferromagnetic particles having a particle size between 10 and 60 microns. Such particles may, for example, contain a core of mica and magnetic particles immobilized thereon, which is enclosed by the glass layer. While in WO 96/4181 the magnetic particles are presented in solid form, for example as tablets or powders, in the respective reaction vessels used, the magnetic particles are used according to the invention in the form of an alcoholic suspension. Particularly suitable are alcoholic suspensions with a concentration of about 5 to 20 mg / ml proved. Surprisingly, it was found that, despite the high specific gravity of the magnetic glass particles, the suspension can be withdrawn from a storage container with high reproducibility, which makes it possible to automate the process.

Obwohl beim erfindungsgemäßen Verfahren die in WO 96/41811 beschriebenen Glaspartikel gute Resultate zeigen, werden besonders gute Ergebnisse mit Glaspartikeln erhalten, deren Glasphase folgende Metalloxide umfasst. SiO2, B2O3, Alkalimetalloxid, z.B. K2O oder/und Na2O sowie gegebenenfalls Al2O3 und ein Erdalkalimetalloxid z.B. CaO. Die Anteile dieser Metalloxide sind vorzugsweise wie folgt: 50 bis 95 mol-% SiO2, 0,2 bis 30 mol-% B2O3, 0 bis 10 mol-% Al2O3, 0 bis 20 mol-% Erdalkalimetalloxid und 0,2 bis 20 mol-% Alkalimetalloxid, wobei die Prozentangaben jeweils auf das Gesamtgewicht der Glasphase bezogen sind.Although in the method according to the invention in WO 96/41811 glass particles described show good results, particularly good results are obtained with glass particles whose glass phase comprises the following metal oxides. SiO 2 , B 2 O 3 , alkali metal oxide, for example K 2 O or / and Na 2 O and optionally Al 2 O 3 and an alkaline earth metal oxide, for example CaO. The proportions of these metal oxides are preferably as follows: 50 to 95 mol% SiO 2 , 0.2 to 30 mol% B 2 O 3 , 0 to 10 mol% Al 2 O 3 , 0 to 20 mol% alkaline earth metal oxide and 0.2 to 20 mol% alkali metal oxide, wherein the percentages are based on the total weight of the glass phase.

Für die Isolierung von RNA hat sich beispielsweise eine Glasphase, die SiO2, B2O3, K2O, Al2O3 und CaO enthält, als besonders geeignet erwiesen. Für die Isolierung von DNA hat sich eine Glasphase, die SiO2, B2O3 und Na2O enthält, als besonders geeignet erwiesen.For the isolation of RNA, for example, a glass phase containing SiO 2 , B 2 O 3 , K 2 O, Al 2 O 3 and CaO has proven to be particularly suitable. For the isolation of DNA, a glass phase containing SiO 2 , B 2 O 3 and Na 2 O proved to be particularly suitable.

Die Adsorptionsmatrix wird beim erfindungsgemäßen Verfahren vorzugsweise in einer Menge zugegeben, welche der minimalen, zur quantitativen Bindung des in der Probe vorhandenen Analyten, insbesondere einer Nukleinsäure, benötigten Menge entspricht oder etwas größer, vorzugsweise um höchstens 50% und besonders bevorzugt um höchstens 20% über dieser Menge liegt. Die zu erwartende Nukleinsäuremenge in verschiedenen Arten von Proben kann - sofern sie nicht bereits bekannt ist - vorab durch übliche Techniken, z.B. Phenol/ Chloroform-Extraktion und anschließende Messung der optischen Dichte ermittelt werden.In the method according to the invention, the adsorption matrix is preferably added in an amount which corresponds to the minimum amount required for quantitatively binding the analyte present in the sample, in particular a nucleic acid, or slightly larger, preferably not more than 50% and particularly preferably not more than 20% this amount is. The expected amount of nucleic acid in different types of samples, unless already known, may be determined in advance by conventional techniques, e.g. Phenol / chloroform extraction and subsequent measurement of the optical density can be determined.

Schritt (a) des erfindungsgemäßen Verfahrens umfaßt das Aufschließen der Probe in einem Reaktionsgefäß. Dieser Aufschluß erfolgt im Allgemeinen durch Lyse der in der Probe vorhandene Zellen unter denaturierenden Bedingungn ,z.B. durch Zugabe einer Protease und eines Denaturierungspuffers. Als Proteinase wird vorzugsweise Proteinase K, Pronase, Elastase oder/und Lysozym verwendet. Besonders bevorzugt ist die Verwendung von Proteinase K.Step (a) of the process of the invention comprises digesting the sample in a reaction vessel. This digestion is generally carried out by lysing the cells present in the sample under denaturing conditions, e.g. by adding a protease and a denaturation buffer. The proteinase used is preferably proteinase K, pronase, elastase or / and lysozyme. Particularly preferred is the use of proteinase K.

Der Proteaseverdau erfolgt in einem Denaturierungspuffer, der eine chaotrope Verbindung, z.B. Harnstoff oder Harnstoffderivate, vorzugsweise ein chaotropes Salz, besonders bevorzugt ein Guanidiniumsalz wie etwa Guanidiniumhydrochlorid (insbesondere zur Isolierung von DNA) oder Guanidiniumthiocyanat (insbesondere zur Isolierung von RNA) oder ein Perchlorat oder Iodid enthält. Für Guanidiniumsalze sind Konzentrationen im Bereich von 1 bis 3 mol/l bevorzugt.Protease digestion is carried out in a denaturation buffer containing a chaotropic compound, e.g. Urea or urea derivatives, preferably a chaotropic salt, more preferably a guanidinium salt such as guanidinium hydrochloride (in particular for the isolation of DNA) or guanidinium thiocyanate (in particular for the isolation of RNA) or a perchlorate or iodide. For guanidinium salts, concentrations in the range of 1 to 3 mol / l are preferred.

Im Gegensatz zu der in WO 96/41811 beschriebenen Methode zur Probenvorbereitung erfolgt die Zugabe der festen Adsorptionsmatrix erst nach Aufschluß der Probe. Durch diese Verfahrensführung erreicht man eine signifikant geringere unspezifische Bindung von unerwünschten Probenbestandteilen, z.B. Proteinen, an der Adsorptionsmatrix.Unlike the in WO 96/41811 described method for sample preparation, the addition of the solid adsorption matrix takes place only after digestion of the sample. This process procedure achieves a significantly lower unspecific binding of undesired sample constituents, for example proteins, to the adsorption matrix.

Gemäß Schritt (c) erfolgt die selektive Bindung des Analyten an die Adsorptionsmatrix durch Inkubation im Aufschlußpuffer vorzugsweise unter chaotropen Bedingungen.According to step (c), the selective binding of the analyte to the adsorption matrix is carried out by incubation in the digestion buffer, preferably under chaotropic conditions.

Schritt (d) des erfindungsgemäßen Verfahrens umfaßt das Abtrennen nicht gebundener Probenbestandteile von der Adsorptionsmatrix. Vorzugsweise werden hierzu die nichtgebundenen Probenbestandteile aus dem Reaktiosgefäß entfernt. Dies kann durch gegebenenfalls mehrmaliges Zugeben und Entfernen eines Waschpuffers erfolgen, der vorzugsweise einen Gehalt von mindestens 50% (v/v) und besonders bevorzugt von mindestens 60% (v/v) eines mit Wasser mischbaren organischen Lösungsmittels wie etwa Ethanol, Propanol und Aceton enthält.Step (d) of the method of the invention comprises separating unbound sample components from the adsorption matrix. Preferably, for this purpose, the unbound sample components are removed from the reactor vessel. This can be done by optionally adding and removing a washing buffer, preferably containing at least 50% (v / v) and more preferably at least 60% (v / v) of a water-miscible organic solvent such as ethanol, propanol and acetone contains.

Die Schritte (c), (d) oder/und (e) des erfindungsgemäßen Verfahren erfolgen vorzugsweise unter kontinuierlichem oder intervallartigem Mischen (d.h. Phasen, während denen gemischt wird, wechseln sich mit Phasen ab, in denen sich das Reaktionsgefäß im Ruhen befindet) ohne Zusatz externer Mittel. Vorzugsweise erfolgt dieses Mischen durch Drehung des Reaktionsgefäßes um seine Längsachse mit mehrmaliger Umkehr der Drehrichtung. Besonders bevorzugt wird das Mischungsgefäß exakt um seine Längsachse gedreht und der Drehrichtungswechsel so durchgeführt, daß die Meniskusauslenkung der Flüssigkeit unter einer vorbestimmten Trennziffer bleibt. Solche Mischverfahren sind in WO91/15768 und EP-A-0 435 481 beschrieben.The steps (c), (d) or / and (e) of the process according to the invention are preferably carried out with continuous or interval-like mixing (ie phases during which mixing takes place with phases in which the reaction vessel is at rest) without Addition of external funds. This mixing preferably takes place by rotation of the reaction vessel about its longitudinal axis with repeated reversal of the direction of rotation. Particularly preferably, the mixing vessel is rotated exactly about its longitudinal axis and the change of direction is performed so that the Meniskusauslenkung the liquid remains below a predetermined separation number. Such mixing methods are in WO91 / 15768 and EP-A-0 435 481 described.

Die Dauer für die Schritte (c) oder/und (e) beträgt vorzugsweise maximal 20 min und umfaßt ein kontinuierliches Mischen oder ein Intervallmischen in kurzen Zyklen, vorzugsweise in kurzen Zyklen von vorzugsweise maximal 2 Minuten. Besonders gute Ergebnisse wurden durch Intervallmischen in einem einminütigen Zyklus umfassend 20 sec Mischen und 40 sec Ruhen erhalten.The duration for steps (c) or / and (e) is preferably at most 20 minutes and comprises continuous mixing or interval mixing in short cycles, preferably in short cycles of preferably maximally 2 minutes. Particularly good results were obtained by interval mixing in a one-minute cycle comprising 20 sec of mixing and 40 sec of rest.

Bei Verwendung von magnetischen Partikeln als Adsorptionsmatrix kann die Zugabe von Flüssigkeiten in das Reaktionsgefäß bzw. das Absaugen von Flüssigkeiten daraus unter kontinuierlichem Mischen erfolgen, wobei die Partikel während des Absaugevorgangs durch Einschalten des Magneten im Reaktionsgefäß gehalten werden. Durch diese Mischtechnik kann das erfindungsgemäße Verfahren flexibel auf verschiedene Probenarten eigestellt werden. Darüber hinaus wird ständig für eine gleichmäßige Verteilung der magnetischen Partikel in der Flüssigphase gesorgt.When using magnetic particles as the adsorption matrix, the addition of liquids into the reaction vessel or the aspiration of liquids from it can be carried out with continuous mixing, wherein the particles are held in the reaction vessel during the suction by switching on the magnet. By means of this mixing technique, the method according to the invention can be flexibly adjusted to different types of samples. In addition, a constant distribution of the magnetic particles in the liquid phase is constantly ensured.

Schritt (e) des erfindungsgemäßen Verfahrens umfasst die Elution des Analyten von der Adsorptionsmatrix. Hierzu kann einerseits - wie aus dem Stand der Technik bekannt - ein von organischen Lösungsmitteln im wesentlichen freier Niedrigsalzpuffer verwendet werden. Überraschenderweise wurde jedoch festgestellt, daß der Elutionspuffer zusätzliche Reagenzien enthalten kann, wie etwa Enzyme, z.B. zur Manipulation von Nukleinsäuren verwendete Enzyme wie etwa RNasen, DNasen, Restriktionsendonukleasen, Ligasen, terminale Transferasen oder/und Polymerasen. Wenn der Analyt beispielsweise eine DNA ist, kann während der Elution eine DNase-freie RNase zugesetzt werden, um den Gehalt an unerwünschter RNA zu verringern. Andererseits kann - wenn der Analyt eine RNA ist - während der Elution eine RNase-freie DNase zugesetzt werden. Auf entsprechende Weise können auch andere Enzyme, wie etwa Restriktionsendonukleasen, etc. zugesetzt werden. Wenn die durch das erfindungsgemäße Verfahren isolierte Nukleinsäure nachfolgend einer Amplifikation unterzogen wird, kann während der Elution auch ein Nukleinsäureamplifikations-Mastermix, welcher den Amplifikationspuffer, Nukleotide, Primer, Polymerase und Puffersalze enthält, zugesetzt werden.Step (e) of the method according to the invention comprises the elution of the analyte from the adsorption matrix. For this purpose, on the one hand, as known from the prior art, a low-salt buffer substantially free of organic solvents can be used. Surprisingly, however, it has been found that the elution buffer may contain additional reagents, such as enzymes, e.g. enzymes used to manipulate nucleic acids, such as RNases, DNases, restriction endonucleases, ligases, terminal transferases and / or polymerases. For example, if the analyte is a DNA, DNase-free RNase may be added during elution to reduce the level of unwanted RNA. On the other hand, if the analyte is an RNA, an RNase-free DNase can be added during elution. Similarly, other enzymes such as restriction endonucleases, etc. may also be added. When the nucleic acid isolated by the method of the invention is subsequently subjected to amplification, a nucleic acid amplification master mix containing the amplification buffer, nucleotides, primers, polymerase and buffer salts may also be added during the elution.

Schritt (f) des erfindungsgemäßen Verfahrens umfaßt das Abtrennen des Eluats von der Adsorptionsmatrix. Dieses Abtrennen kann auf übliche Weise erfolgen, z.B. durch Sedimentation, vorzugsweise aber durch magnetische Separation.Step (f) of the process of the invention comprises separating the eluate from the adsorption matrix. This separation can be carried out in the usual way, for example by sedimentation, but preferably by magnetic separation.

Die durch das erfindungsgemäße Verfahren isolierten Analyten können anschließend auf bekannte Weise weiterverarbeitet werden, im Fall von Nukleinsäuren, z.B. durch Amplifikation und nachfolgende Detektion, Detektion ohne vorhergehende Amplifikation oder Sequenzierung. Hierzu können Aliquots des Eluats selbst Bestimmungen unterschiedlicher Analyten zugeführt werden, z.B. verschiedene Viren, wie HIV, HCV und HBV.The analytes isolated by the method according to the invention can then be further processed in a known manner, in the case of nucleic acids, e.g. by amplification and subsequent detection, detection without prior amplification or sequencing. For this purpose, aliquots of the eluate itself may be supplied to determinations of different analytes, e.g. various viruses, such as HIV, HCV and HBV.

Ein wichtiges Merkmal des erfindungsgemäßen Verfahrens ist, daß viele oder gegebenenfalls sogar alle Schritte bei im wesentlichen der gleichen Temperatur, d.h. innerhalb eines Temperaturbereichs von ± 2,5°C durchgeführt werden können. Vorzugsweise ist diese Temperatur im Bereich von Raumtemperatur bis 70°C, besonders vorzugt von Raumtemperatur bis 40°C, am meisten bevorzugt bei Raumtemperatur, d.h. ca. 18 bis 32°C. In einer bevorzugten Ausführungsform des erfindungsgemäßen Verfahrens werden zumindest die Schritte (c) der Adsorption und (d) des Waschens bei dieser Temperatur durchgeführt. Besonders bevorzugt werden auch andere Schritte, insbesondere die Schritte (a) des Aufschließens oder/und (e) der Elution bei dieser Temperatur durchgeführt. Für die Bestimmung von HIV in Blutproben kann beispielsweise die gesamte Probenvorbereitung bei einer einheitlichen Temperatur erfolgen. Gegebenenfalls kann nach Schritt (f) des erfindungsgemäßen Verfahrens zusätzlich ein Nachbehandlungsschritt bei erhöhter Temperatur erfolgen, wodurch bei bestimmten Analyten die Ausbeuten bei einer Amplifikation verbessert wird. Bei anderen Analyten kann es erforderlich sein, daß die Vorbehandlung oder/und die Elution bei einer erhöhte Temperatur erfolgen. Die erhöhte Temperatur liegt dabei vorzugsweise im Bereich von mehr als 40°C bis 95°C, z.b. ca. 70 °C.An important feature of the process of the invention is that many, or even all, of the steps are conducted at substantially the same temperature, i. within a temperature range of ± 2.5 ° C. Preferably, this temperature is in the range of room temperature to 70 ° C, more preferably from room temperature to 40 ° C, most preferably at room temperature, i. about 18 to 32 ° C. In a preferred embodiment of the method according to the invention, at least the steps (c) of the adsorption and (d) of the washing are carried out at this temperature. Particular preference is also given to carrying out other steps, in particular steps (a) of digesting and / or (e) elution at this temperature. For example, for the determination of HIV in blood samples, the entire sample preparation may be done at a uniform temperature. Optionally, after step (f) of the process according to the invention, an additional post-treatment step may be carried out at elevated temperature, whereby the yields in the case of certain analytes are improved during an amplification. Other analytes may require pretreatment and / or elution at elevated temperature. The elevated temperature is preferably in the range of more than 40 ° C to 95 ° C, z.b. about 70 ° C.

Die Durchführung des erfindungsgemäßen Verfahrens erfolgt vorzugsweise in einer automatisierten Vorrichtung. Beispiele für solche Vorrichtungen sind nachfolgend beschrieben. Weiterhin ist bevorzugt, daß das erfindungsgemäße Verfahren zur Probenvorbereitung, zumindest die Schritte (a) bis (e) in einem einzigen Reaktionsgefäß durchgeführt werden, d.h. daß kein Transfer in ein anderes Reaktionsgefäß erfolgt. Dies hat eine erhebliche Vereinfachung des Verfahrens zur Folge und führt darüber hinaus zu einem verringertem Kontaminationsrisiko.The implementation of the method according to the invention is preferably carried out in an automated device. Examples of such devices are described below. Furthermore, it is preferred that the inventive A method for sample preparation, at least steps (a) to (e) are carried out in a single reaction vessel, ie that no transfer to another reaction vessel takes place. This results in a considerable simplification of the method and also leads to a reduced risk of contamination.

Noch ein weiterer Gegenstand der Erfindung ist ein Reagenzienkit, der insbesondere zur Durchführung des vorstehend beschriebenen Verfahrens geeignet ist, umfassend

  1. (a) eine Protease,
  2. (b) einen Probenaufschlußpuffer,
  3. (c) einen Waschpuffer,
  4. (d) einen Elutionspuffer und
  5. (e) eine alkoholische Suspension von magnetischen Glaspartikeln.
Yet another object of the invention is a reagent kit which is particularly suitable for carrying out the method described above, comprising
  1. (a) a protease,
  2. (b) a sample digest buffer,
  3. (c) a wash buffer,
  4. (d) an elution buffer and
  5. (e) an alcoholic suspension of magnetic glass particles.

Auch beschrieben, aber nicht beansprucht, ist eine Vorrichtung zur Isolierung eines Analyten aus einer biologischen Probe, umfassend:

  • eine Aufnahmeeinrichtung für Reagenzien (2),
  • eine erste Aufnahmeeinrichtung für Reaktionsgefäße zur Probenvorbereitung (3), die für eine Betriebstemperatur von ≤ 70°C, insbesondere ≤ 40°C eingerichtet ist,
  • eine zweite Aufnahmeeinrichtung für Reaktionsgefäße (4a, 4b, 4c), die gegebenenfalls Kühl- oder/und Heizmittel enthält,
  • und automatische Pipettiervorrichtungen.
Also described but not claimed is a device for isolating an analyte from a biological sample comprising:
  • a receiving device for reagents (2),
  • a first receiving device for reaction vessels for sample preparation (3), which is set up for an operating temperature of ≤ 70 ° C, in particular ≤ 40 ° C,
  • a second receptacle for reaction vessels (4a, 4b, 4c), which optionally contains cooling and / or heating means,
  • and automatic pipetting devices.

Die Vorrichtung ist vorzugsweise so ausgestaltet, daß ein einziges Reaktionsgefäß zur Durchführung der 4 Hauptschritte der Probenvorbereitung, nämlich Aufschluß einer Probe bzw. Lyse, Adsorption des freigesetzten Analyten, z.B. einer Nukleinsäure, an eine feste Adsorptionsmatrix, z.B. magnetische Glaspartikel, Waschen der Adsorptionsmatrix und Elution des Analyten von der Adsorptionsmatrix vorgesehen ist.The apparatus is preferably designed so that a single reaction vessel for carrying out the four main steps of sample preparation, namely digestion of a sample or lysis, adsorption of the released analyte, e.g. a nucleic acid, to a solid adsorption matrix, e.g. magnetic glass particles, washing the adsorption matrix and elution of the analyte is provided by the adsorption matrix.

Die Vorrichtung ist so ausgestaltet, daß die erste Aufnahmeeinrichtung zur Aufnahme der Reaktionsgefäße für die Probenvorbereitung zumindest für die Adsorption des Analyten an die fest Adsorptionsmatrix und für das Waschen der Adsorptionsmatrix vorgesehen ist. In einer bevorzugten Ausführungsform ist die erste Aufnahmeeinrichtung weiterhin zum Aufschluß der Probe oder/und zur Elution des Analyten von der Adsorptionsmatrix vorgesehen. Die Reaktionsgefäße zur Probenvorbereitung haben ein Volumen von vorzugsweise mindestens 1 ml, z.B. 1-5 ml.The device is designed so that the first receiving device for receiving the reaction vessels for sample preparation is provided at least for the adsorption of the analyte to the solid adsorption matrix and for washing the adsorption matrix. In a preferred embodiment, the first receiving device is further provided for digesting the sample and / or for eluting the analyte from the adsorption matrix. The reaction vessels for sample preparation have a volume of preferably at least 1 ml, e.g. 1-5 ml.

Die zweite Aufnahmeeinrichtung ist für Reaktionsgefäße zur Aufbewahrung oder/und Weiterverarbeitung des Analyten vorgesehen, z.B. PCR-Gefäße, die üblicherweise eine von den zur Probenvorbereitung verwendeten Reaktionsgefäßen verschiedene Form aufweisen. Die Reaktionsgefäße zur Aufbewahrung oder/und Weiterverarbeitung haben ein Volumen von vorzugsweise bis zu 500 µl, z.B. 50-200 µl. Darüber hinaus kann die zweite Aufnahmeeinrichtung Gefäße für Reagenzien enthalten, die zur Weiterverarbeitung der den Analyten enthaltenden Probe benötigt werden, z.B. einen PCR-Mastermix.The second receptacle is intended for reaction vessels for storage or / and further processing of the analyte, e.g. PCR vessels, which usually have a different shape from the reaction vessels used for sample preparation. The reaction vessels for storage and / or further processing have a volume of preferably up to 500 μl, e.g. 50-200 μl. In addition, the second receptacle may contain vessels for reagents needed for further processing of the sample containing the analyte, e.g. a PCR master mix.

Die Vorrichtung kann so ausgestaltet sein, daß einer oder mehrere Schritte der Probenvorbereitung oder/und ein Nachbehandlungsschritt bei einer erhöhten Temperatur in der zweiten Aufnahmeeinrichtung erfolgen können. Hierzu kann die zweite Aufnahmeeinrichtung zur Aufnahme von Reaktionsgefäßen für zumindest einen Behandlungsschritt vorgesehen sein, der ausgewählt ist aus dem Aufschluß der Probe, der Elution der Probe von der Adsorptionsmatrix und einem Nachbehandlungsschritt nach der Elution.The apparatus may be configured such that one or more steps of sample preparation and / or an aftertreatment step may be performed at an elevated temperature in the second receiving means. For this purpose, the second receiving device may be provided for receiving reaction vessels for at least one treatment step, which is selected from the digestion of the sample, the Elution of the sample from the adsorption matrix and post-treatment step after elution.

Die erste Aufnahmeeinrichtung umfaßt vorzugsweise Mittel zur magnetischen Separation. Weiterhin ist bevorzugt, daß die erste Aufnahmeeinrichtung Mittel zum Mischen der Reaktionsgefäße, insbesondere durch Drehen um deren Längsachse umfaßt. Solche Mittel können gegebenenfalls auch für die zweite Aufnahmeeinrichtung vorgesehen sein.The first receiving device preferably comprises means for magnetic separation. Furthermore, it is preferred that the first receiving means comprises means for mixing the reaction vessels, in particular by rotating about their longitudinal axis. If appropriate, such means can also be provided for the second receiving device.

Die Vorrichtung umfasst im allgemeinen automatische Pipettiereinrichtungen sowie gegebenenfalls Mittel zum Transport von Reaktionsgefäßen, z. B. zwischen erster und zweiter Aufnahmeeinrichtung. Außerdem kann eine Deckel-Öffnungs- und Schließeinheit integriert sein.The device generally comprises automatic pipetting devices and optionally means for transporting reaction vessels, e.g. B. between first and second receiving device. In addition, a lid opening and closing unit can be integrated.

Im folgenden sind spezielle Ausführungsformen im Detail dargestellt. Bei der in Abbildung 1 gezeigten Ausführungsform enthält die Vorrichtung (1) eine Aufnahmeeinrichtung für Reagenzien (2), eine Aufnahmeeinrichtung für Reaktionsgefäße zur Probenvorbereitung (3) mit den Funktionen Mischen und Magnetseparation, die für eine Temperatur von vorzugsweise ≤ 40°C und besonders bevorzugt Raumtemperatur vorgesehen ist. Weiterhin enthält die Vorrichtung eine Aufnahmestation für weitere Reaktionsgefäße (4a), z.B. für PCR-Gefäße, die eine Temperatur von 4°C bis Raumtemperatur aufweist. Weiterhin enthält die Vorrichtung automatisierte Einrichtungen für die Pipettierung und Handhabung von Reaktionsgefäßen (5), die Bewegungen in X, Y und Z Richtung ermöglichen. Bei dieser Ausführungsform der erfindungsgemäßen Vorrichtung finden die vier Hauptschritte der Probenvorbereitung, nämlich Lyse, Adsorption, Waschen und Elution in der ersten Aufnahmeeinrichtung in einem einzigen Reaktionsgefäß statt. Die Lagerung von Eluaten und die Zugabe weiterer Reagenzien, z.B. PCR-Mastermix, erfolgt in der zweiten Aufnahmeeinrichtung. Zur Weiterverarbeitung, z.B. für eine nachfolgende PCR, werden die Gefäße zu einer entsprechenden Vorrichtung, z.B. einem Thermocycler (nicht gezeigt) transferiert.In the following special embodiments are shown in detail. In the embodiment shown in FIG. 1, the device (1) contains a receiving device for reagents (2), a receptacle for reaction preparation tubes (3) with the functions of mixing and magnetic separation, preferably for a temperature of ≦ 40 ° C. and more preferably Room temperature is provided. Furthermore, the device contains a receiving station for further reaction vessels (4a), for example for PCR vessels, which has a temperature of 4 ° C to room temperature. Furthermore, the device contains automated devices for the pipetting and handling of reaction vessels (5), which allow movements in the X, Y and Z directions. In this embodiment of the device according to the invention, the four main steps of sample preparation, namely lysis, adsorption, washing and elution, take place in the first receiving device in a single reaction vessel. The storage of eluates and the addition of further reagents, eg PCR master mix, takes place in the second receiving device. For further processing, eg for a subsequent PCR, the vessels are transferred to a respective device, eg a thermal cycler (not shown).

In der in Abbildung 2 gezeigten Ausführungsform enthält die Vorrichtung eine zweite Aufnahmeeinrichtung (4b), die zur Aufnahme von Weiterverarbeitungsreaktionsgefäßen, z.B PCR-Gefäßen, vorgesehen ist und die Einstellung einer Temperatur von 4°C (Kühlung des PCR-Mastermix) bis 95°C zum Erhitzen des Eluats nach der Elution von der Adsorptionsmatrix vorgesehen ist. Zur Vermeidung der Kondensatbildung am Deckel der PCR-Gefäße ist eine Deckelgegenheizung bevorzugt.In the embodiment shown in Figure 2, the device contains a second receiving device (4b), which is intended to receive further processing reaction vessels, such as PCR vessels, and setting a temperature of 4 ° C (cooling of the PCR master mix) to 95 ° C. for heating the eluate after elution from the adsorption matrix. To avoid the formation of condensation on the lid of the PCR vessels, a lid heater is preferred.

Gemäß der in Abbildung 3 dargestellten Ausführungsform der erfindungsgemäßen Vorrichtung ist eine zweite Aufnahmeeinrichtung für Reaktionsgefäße (4c) vorgesehen, welche zur Aufnahme von PCR-Gefäßen und Probenvorbereitungsgefäßen vorgesehen ist. In dieser zweiten Aufnahmeeinrichtung kann eine Kühlung, z.B. auf 4°C, und ein Aufheizen, z.B. auf 95°C, zum Erhitzen des Lysats oder/und des Eluats erfolgen. Auch hier ist zur Vermeidung der Kondensatbildung am Deckel von Reaktionsgefäßen eine Deckelgegenheizung bevorzugt.According to the embodiment of the device according to the invention shown in FIG. 3, a second receptacle for reaction vessels (4c) is provided, which is provided for receiving PCR vessels and sample preparation vessels. In this second receiving device, cooling, e.g. to 4 ° C, and heating, e.g. to 95 ° C, to heat the lysate and / or the eluate. Again, a lid heater is preferred to avoid the formation of condensation on the lid of reaction vessels.

Gemäß noch einer weiteren Ausführungsform der vorliegenden Erfindung (nicht gezeigt) ist die erste Aufnahmeeinrichtung zur Einstellung einer Temperatur im Bereich von ≤ 70°C vorgesehen. Die zweite Aufnahmeeinrichtung ist - wie in Abbildung 3 gezeigt - für das Kühlen und Heizen von Probenweiterverarbeitungs- und Probenvorbereitungsgefäßen geeignet.According to yet another embodiment of the present invention (not shown), the first receiving means is provided for setting a temperature in the range of ≦ 70 ° C. The second receiver, as shown in Figure 3, is suitable for cooling and heating sample processing and sample preparation vessels.

Weiterhin wird die vorliegende Anmeldung durch die folgenden Figuren und Beispiele näher erläutert. Es zeigen:

Abbildung 1
die schematische Darstellung einer ersten Ausführungsform der Vorrichtung,
Abbildung 2
die schematische Darstellung einer zweiten Ausführungsform der Vorrichtung.
Abbildung 3
die schematische Darstellung einer dritten Ausführungsform der Vorrichtung,
Abbildung 4
das Ergebnis eines Chlamydien-Nachweises durch PCR mit manueller und semiautomatisierter Probenvorbereitung,
Abbildung 5
das Ergebnis eines Chlamydien-Nachweises durch PCR mit semiautomatisierter Probenvorbereitung und unterschiedlichen Temperaturprofilen bei der Probenvorbereitung,
Abbildung 6
das Ergebnis eines HIV-Nachweises durch PCR mit manueller Probenvorbereitung (Standardprotokoll) und semiautomatisierter Probenvorbereitung bei Raumtemperatur.
Furthermore, the present application is explained in more detail by the following figures and examples. Show it:
illustration 1
the schematic representation of a first embodiment of the device,
Figure 2
the schematic representation of a second embodiment of the device.
Figure 3
the schematic representation of a third embodiment of the device,
Figure 4
the result of chlamydia detection by PCR with manual and semi-automated sample preparation,
Figure 5
the result of chlamydia detection by PCR with semi-automated sample preparation and different temperature profiles during sample preparation,
Figure 6
the result of HIV detection by PCR with manual sample preparation (standard protocol) and semi-automated sample preparation at room temperature.

BeispieleExamples 1. Herstellung von magnetischen Glaspartikeln1. Production of magnetic glass particles

Es wurden zwei verschiedene Sole verwendet. Die Herstellung der Sole erfolgte wie folgt:Two different sols were used. The preparation of the brine was as follows:

Sol 1: (SiOSol 1: (SiO 22 :B: B 22 OO 33 :Na:N / A 22 O=40:8:2)O = 40: 8: 2)

Alkokolate der Oxide wurden in obigen Molverhältnissen analog der Vorgehensweise in Beispiel 1 und 2 von W096/4181 miteinander zu einer homogenen Phase verrührt. In Abweichung dazu wurde kein HCl eingesetzt.Alkokolates of the oxides were in the above molar ratios analogous to the procedure in Example 1 and 2 of W096 / 4181 stirred together to a homogeneous phase. By contrast, no HCl was used.

Anschließend wurden 30 g Iriodin 600 Black Mica (Merk) in 100 ml Sol eingerührt.Subsequently, 30 g of Iriodin 600 Black Mica (Merk) were stirred into 100 ml of sol.

Sol 2: (SiOSol 2: (SiO 22 :B: B 22 OO 33 :K: K 22 O:AlO: Al 22 OO 33 :CaO=76:15:5:2:2): CaO = 76: 15: 5: 2: 2)

Alkokolate der Oxide wurden in obigen Molverhältnissen analog der Vorgehensweise in Beispiel 1 und 2 von WO96/41811 miteinander zu einer homogenen Phase verrührt. In Abweichung dazu wurde kein HCl eingesetzt.Alkokolates of the oxides were in the above molar ratios analogous to the procedure in Example 1 and 2 of WO96 / 41811 stirred together to a homogeneous phase. By contrast, no HCl was used.

Anschließend wurden 30 g Iriodin 600 Black Mica (Merk) in 100 ml Sol eingerührt.Subsequently, 30 g of Iriodin 600 Black Mica (Merk) were stirred into 100 ml of sol.

Die Sole wurden anschließend einem Sprühtrocknungsvorgang unterzogen.The sols were then subjected to a spray-drying process.

Das durch die Sprütrocknung erhaltene Pulver wurde einer Feinteilabtrennung durch Sedimentation, einer Temperaturbehandlung unter Stickstoffatmosphäre (60 l/h Volumenstrom) bei einer Aufheizgeschwindigkeit von 1 K/min unterzogen und 1 h einer Verdichtungstemperatur im Bereich von 600 bis 700°C für eine Stunde gehalten. Anschließend wurde der Ofen auf 300°C abgekühlt und bei dieser Temperatur für 1 h mit Sauerstoff gespült. Nach Abkühlung auf Raumtemperatur wurden die magnetischen Glaspartikel entnommen und zur Abtrennung des Grobanteils auf ein 50 µm-Sieb aufgegeben und gesiebt.The powder obtained by the desiccation was subjected to fine particle separation by sedimentation, temperature treatment under nitrogen atmosphere (60 l / h volume flow) at a heating rate of 1 K / min, and held for 1 hour at a compression temperature in the range of 600 to 700 ° C for one hour. Subsequently, the furnace was cooled to 300 ° C and rinsed at this temperature for 1 h with oxygen. After cooling to room temperature, the magnetic glass particles were removed and placed on a 50 micron sieve and separated to separate the coarse fraction.

Die aus Sol 1 erhaltenen magnetischen Glaspartikel sind insbesondere zur Isolierung von DNA geeignet. Die aus Sol 2 erhaltenen Glaspartikel sind insbesondere zur Isolierung von RNA geeignet.The magnetic glass particles obtained from sol 1 are particularly suitable for the isolation of DNA. The glass particles obtained from sol 2 are particularly suitable for the isolation of RNA.

2. Standardprotokoll zur Probenvorbereitung für die Isolierung von Nukleinsäuren, z.B. DNA2. Standard protocol for sample preparation for the isolation of nucleic acids, e.g. DNA

Zur Isolierung von Nukleinsäuren aus biologischen Proben wie etwa Vollblut oder kultivierten Zellen ist das folgende Standardprotokoll geeignet. Die auf diese Weise erhaltenen Nukleinsäuren können direkt nach der Elution für eine Amplifikation durch PCR, eine Restriktionsspaltung oder einen Southernblot eingesetzt werden.For isolating nucleic acids from biological samples such as whole blood or cultured cells, the following standard protocol is suitable. The nucleic acids obtained in this way can be used immediately after elution for an amplification by PCR, a restriction cleavage or a Southern blot can be used.

Der Reaktionskit enthält:

  1. 1. Bindepuffer (4,7 mol/l Guanidiniumhydrochlorid, 10mmol/l Harnstoff, 10 mmol/l Tris HCl, 20% Triton® X-100, pH 5,7
  2. 2. lyophilisierte Proteinase K (zur Auflösung in H2O auf eine Konzentration von 20 mg/ml)
  3. 3. Waschpuffer (56% (v/v) Ethanol, 20 mmol/NaCl, 10 mmol/l Tris HCl pH 7,5)
  4. 4. Elutionspuffer (10 mmol/l Tris pH 8,5)
  5. 5. magnetische Glaspartikel (MPG)
    1. a) Tabletten mit jeweils 7,5 mg der Glaspartikel oder
    2. b) 15%ige Suspension der Glaspartikel in Ethanol
The reaction kit contains:
  1. 1. Binding buffer (4.7 mol / l guanidinium hydrochloride, 10 mmol / l urea, 10 mmol / l Tris HCl, 20% Triton® X-100, pH 5.7
  2. 2. lyophilized proteinase K (for dissolution in H 2 O to a concentration of 20 mg / ml)
  3. 3. Wash Buffer (56% (v / v) ethanol, 20 mmol / NaCl, 10 mmol / l Tris HCl pH 7.5)
  4. 4. Elution buffer (10 mmol / l Tris pH 8.5)
  5. 5. magnetic glass particles (MPG)
    1. a) tablets each containing 7.5 mg of the glass particles or
    2. b) 15% suspension of the glass particles in ethanol

Die Kitkomponenten sind stabil und können bei Raumtemperatur gelagert werden. Nach Auflösung der Proteinase K in Wasser sollte die Lösung aliquotiert und bei -20°C aufbewahrt werden. Die eingefrorene Lösung ist für 12 Monate stabil.The kit components are stable and can be stored at room temperature. After dissolution of proteinase K in water, the solution should be aliquoted and stored at -20 ° C. The frozen solution is stable for 12 months.

Standardprotokollstandard protocol

  1. 1. 200 µl Probe werden in ein 2 ml Reaktionsgefäß gegeben und mit 200 µl Bindepuffer und 40 µl Proteinase K-Lösung versetzt. Anschließend wird für 10 min inkubiert. Die Inkubation erfolgt vorzugsweise bei Raumtemperatur. Unter bestimmten Umständen kann die Inkubationstemperatur jedoch auch auf bis zu 70°C erhöht werden.1. 200 .mu.l of sample are placed in a 2 ml reaction vessel and mixed with 200 .mu.l of binding buffer and 40 .mu.l proteinase K solution. It is then incubated for 10 min. The incubation is preferably carried out at room temperature. However, under certain circumstances, the incubation temperature may be increased up to 70 ° C.
  2. 2. Nach der Inkubation werden 200 µl Isopropanol und eine MGP Tablette (oder alternativ 200 µl MGP Suspension) zugegeben und für 5 min bei Raumtemperatur inkubiert.2. After incubation, 200 μl of isopropanol and one MGP tablet (or alternatively 200 μl of MGP suspension) are added and incubated for 5 min at room temperature.
  3. 3. Das Reaktionsgefäß wird in einen Magnetpartikelseparator (Boehringer Mannheim, Kat. No. 1 641 794) gegeben und für etwa 1 min separiert.3. The reaction vessel is placed in a magnetic particle separator (Boehringer Mannheim, Cat. No. 1 641 794) and separated for about 1 minute.
  4. 4. Der Überstand wird verworfen und die Reaktionsgefäße werden aus dem MP-Seperator entnommen.4. The supernatant is discarded and the reaction tubes are removed from the MP Seperator.
  5. 5. Nach Zugabe von 500 µl Waschpuffer wird der Inhalt des Reaktionsgefäßes gemischt und erneut in den MP-Separator für etwa 1 min gegeben.5. After adding 500 μl wash buffer, mix the contents of the reaction vessel and place again in the MP Separator for about 1 min.
  6. 6. Der Überstand wird verworfen. Schritt 5 wird dreimal widerholt. Nach dem letzten Waschvorgang wird der restliche Waschpuffer vollständig entfernt.6. The supernatant is discarded. Step 5 is repeated three times. After the last wash, the remaining wash buffer is completely removed.
  7. 7. Zur Elution werden 100 µl gegebenenfalls auf 70°C vorgewärmter Elutionspuffer zugegeben. Dann wird gemischt und für 5 Minuten bei Raumtemperatur inkubiert. Die Probe wird in den MP-Separator gegeben und der Überstand in ein sauberes Reaktionsgefäß überführt.7. For elution, 100 μl of elution buffer, optionally prewarmed to 70 ° C., are added. Then it is mixed and incubated for 5 minutes at room temperature. The sample is placed in the MP separator and the supernatant transferred to a clean reaction vessel.
  8. 8. Die so erhaltenen Nukleinsäuren, z.B. DNA, sind stabil und können anschließend direkt weiterverarbeitet oder bei 4°C aufbewahrt werden.8. The nucleic acids thus obtained, e.g. DNA, are stable and can then be further processed directly or stored at 4 ° C.

Das obige Protokoll kann auch entsprechend bei Mikrotiterplatten, z.B. Tieflochmikrotiterplatten (z.B. Ritter, H.J. Bioanalytic), verwendet werden.The above protocol may also be used in accordance with microtiter plates, eg Tieflochmikrotiterplatten (eg Ritter, HJ Bioanalytic).

3. Chlamydia trachomates DNA-Nachweis durch PCR3. Chlamydia trachomates DNA detection by PCR 3.1 Manuelles Standardprotokoll zur Probenvorbereitung3.1 Manual standard protocol for sample preparation

200 µl einer Urinprobe und 240 µl Bindepuffer/Proteinase K-Lösung (5:1) werden in ein 2 ml Reaktionsgefäß pipettiert, einem Vortexmischen unterzogen und bei 70°C für 10 min inkubiert. Dann wird die Probe für 5 min auf Raumtemperatur abgekühlt.200 μl of a urine sample and 240 μl of binding buffer / proteinase K solution (5: 1) are pipetted into a 2 ml reaction vessel, vortexed and incubated at 70 ° C. for 10 min. Then the sample is cooled to room temperature for 5 min.

Zur Probe werden 200 µl isopropanolische MGP-Lösung pipettiert. Unmittelbar anschließend erfolgt Vortexmischen. Die Probe wird dann für 15 min auf einem Mischer, z.B. Thermomischer 5436 (Eppendorf), inkubiert.For the sample, 200 μl of isopropanolic MGP solution are pipetted. Immediately afterwards, vortex mixing takes place. The sample is then placed on a mixer for 15 minutes, e.g. Thermomixer 5436 (Eppendorf), incubated.

Die MGP werden durch Überführung der Probe in einen Magnetseparator konzentriert. Nach einer Minute wird der Überstand vollständig abpipettiert.The MGPs are concentrated by transferring the sample to a magnetic separator. After one minute, the supernatant is completely pipetted off.

Es werden 0,5 ml Waschpuffer zu den MGPs pipettiert. Die Probe wird einem Vortexmischen unterzogen und dann in den Magnetseparator überführt. Der Überstand wird nach 1 min abpipettiert. Die Waschprozedur wird noch zweimal wiederholt.0.5 ml wash buffer is pipetted to the MGPs. The sample is vortexed and then transferred to the magnetic separator. The supernatant is pipetted off after 1 min. The washing procedure is repeated twice more.

Den MGP werden 200 ml Elutionspuffer zugesetzt. Die Probe wird 10 min bei 70°C auf einem Thermomischer bei 1400 RPM inkubiert. Kondenswasser wird durch kurze Zentrifugation gesammelt. Die Probe wird in den Magnetseparator überführt und nach 1 min 180 µl Eluat abgenommen. Das Eluat wird in ein neues Reaktionsgefäß pipettiert und bei 4°C (bei einer Aufbewahrungsdauer < 24 h) oder bei - 20°C (bei längerer Aufbewahrungsdauer) aufbewahrt.200 ml of elution buffer are added to the MGP. The sample is incubated for 10 min at 70 ° C on a thermo-mixer at 1400 RPM. Condensation is collected by short centrifugation. The sample is transferred to the magnetic separator and after 1 min 180 ul eluate removed. The eluate is pipetted into a new reaction vessel and stored at 4 ° C (for a storage period <24 h) or at -20 ° C (for a longer storage period).

Für die PCR werden 50 µl Eluat eingesetzt. Die Auswertung erfolgt durch Elektrochemilumineszenz.For the PCR, 50 μl of eluate are used. The evaluation is carried out by electrochemiluminescence.

3.2 Protokoll für ein semiautomatisiertes Verfahren3.2 Protocol for a semi-automated process

Statt des in 3.1 beschriebenen Vortexmischens und der Temperierung auf einem Thermoblock wird ein semiautomatisiertes Verfahren durchgeführt, bei dem das Mischen und die Temperierung auf einem Misch- und Temperiermodul erfolgt. Abbildung 4 zeigt einen Vergleich der Bestimmung von Chlamydien (Probe: 100 Elementarantikörper pro 100 ml Urin; Sechsfachbestimmung) zwischen dem manuellen Standardprotokoll (Vortex) und dem semiautomatisierten Verfahren (MTM). Es ist ersichtlich, daß durch die Automatisierung keine Beeinträchtigung der Sensitivität erfolgt.Instead of the vortex mixing described in 3.1 and the temperature control on a thermoblock a semi-automated process is carried out, in which the mixing and the temperature control takes place on a mixing and tempering. Figure 4 shows a comparison of the determination of chlamydia (sample: 100 elemental antibodies per 100 ml urine, sixfold determination) between the standard manual protocol (vortex) and the semi-automated method (MTM). It can be seen that the automation does not affect the sensitivity.

3.3 Semiautomatisiertes Protokoll bei Raumtemperatur3.3 Semiautomatized protocol at room temperature

Die Probenvorbereitung erfolgt wie unter Punkt 3.2 beschrieben. Die Lyse und die Elution werden jedoch bei Raumtemperatur durchgeführt.Sample preparation is carried out as described under point 3.2. However, lysis and elution are carried out at room temperature.

3.4 Semiautomatisiertes Probenvorbereitungsprotokoll bei Raumtemperatur mit anschließender Nachbehandlung des Eluats3.4 Semiautomatized sample preparation protocol at room temperature followed by aftertreatment of the eluate

Die Probenvorbereitung erfolgt wie unter Punkt 3.3 beschrieben. Nach der Elution erfolgt eine Inkubation für 10 min bei 70°C.Sample preparation is carried out as described under point 3.3. After elution, incubate for 10 min at 70 ° C.

Abbildung 5 zeigt einen Vergleich der Chlamydienbestimmung (Proben: SWE1, O Chlamydia-Elementarantikörper (EAK) pro ml Urin, SWE 2: 10 EAK, SWE 3:100 EAK und SWE 4:1000 EAK jeweils pro ml Urin) zwischen den in den Punkten 3.2, 3.3 und 3.4 beschriebenen Probenvorbereitungsprotokollen. Es ist ersichtlich, daß für die Bestimmung von Chlamydien das Standardprotokoll gegenüber einer Probenvorbereitung bei Raumtemperatur (RT-Protokoll MTM) sensitiver ist. Die Ergebnisse der Probenvorbereitung bei Raumtemperatur und anschließender Nachbehandlung des Eluats (RT-Protokoll MTM mit Nachbehandlung) zeigen jedoch, daß dieser Effekt größtenteils kompensiert werden kann. Überraschenderweise ist daher während der Probenvorbereitung selbst kein Temperaturschritt erforderlich.Figure 5 shows a comparison of the chlamydia determination (samples: SWE1, O Chlamydia Elemental Antibody (EAC) per mL urine, SWE 2: 10 EAK, SWE 3: 100 EAK and SWE 4: 1000 EAC each per mL urine) between those in the points 3.2, 3.3 and 3.4 described sample preparation protocols. It can be seen that for the determination of Chlamydia the standard protocol is more sensitive to sample preparation at room temperature (RT protocol MTM). However, the results of the sample preparation at room temperature and subsequent treatment of the eluate (RT protocol MTM with post-treatment) show that this effect can be compensated for the most part. Surprisingly, therefore, no temperature step is required during the sample preparation itself.

Durch diese Erkenntnis läßt sich das Probenvorbereitungsverfahren entscheidend vereinfachen, denn die Schritte der Lyse, der Adsorption, des Waschens und der Elution können bei Temperaturen ≤ 40°c erfolgen, was eine Automatisierung vereinfacht, da keine Deckelgegenheizung und Temperaturregulierung erforderlich ist.By this finding, the sample preparation process can be significantly simplified, because the steps of lysis, adsorption, washing and elution can be carried out at temperatures ≤ 40 ° c, which simplifies automation, since no Deckelgegenheizung and temperature regulation is required.

4. HIV-RNA Nachweis durch PCR4. HIV-RNA detection by PCR 4.1 Manuelles Standardprotokoll zur Probenvorbereitung4.1 Manual standard protocol for sample preparation

Geforenes Plasma wird 5 min bei 37°C aufgetaut und zur weiteren Verarbeitung auf Eis gekühlt.Frozen plasma is thawed for 5 min at 37 ° C and cooled on ice for further processing.

In ein 1,5 ml Sarstedt-Reaktionsgefäß werden 50 µl einer Proteinase K Lösung (25 mg/ml) pipettiert. Dazu werden 250 µl Probe gegeben und im Vortex gemischt. Dann werden 300 µl Lysepuffer zugegeben und erneut im Vortex gemischt.50 μl of a proteinase K solution (25 mg / ml) are pipetted into a 1.5 ml Sarstedt reaction vessel. For this purpose, 250 .mu.l sample are added and vortexed. Then 300 μl of lysis buffer are added and vortexed again.

Es wird 10 min bei Raumtemperatur auf einem Eppendorfmischer bei 13.000 RPM inkubiert. Dann werden 300 µl einer MGP Suspension (6 mg/ml MGP in Isopropanol) zugegeben, im Vortex gemischt und 20 min bei Raumtemperatur bei fortgesetztem Mischen inkubiert. Die MGP werden auf einem Magnetseperator abgetrennt und der Überstand vollständig entfernt.It is incubated for 10 min at room temperature on an Eppendorf mixer at 13,000 RPM. Then 300 μl of a MGP suspension (6 mg / ml MGP in isopropanol) are added, vortexed and incubated for 20 minutes at room temperature with continued mixing. The MGP are separated on a Magnetseperator and the supernatant completely removed.

Zu den MGP werden 750 µl Waschpuffer gegeben. Die MGP werden resuspendiert und wie zuvor beschrieben abgetrennt. Die Waschprozedur wird viermal wiederholt, wobei am Ende der Waschpuffer sorgfältig entfernt wird.To the MGP is added 750 μl washing buffer. The MGPs are resuspended and separated as previously described. The washing procedure is repeated four times, at the end of which the washing buffer is carefully removed.

Dann werden 100 µl Elutionspuffer zugegeben und die MGP resuspendiert. Nach 15minütiger Inkubation bei 80°C auf einem Eppendorf Thermomischer (13.000 RPM) werden 90 µl Eluat in eine neues Reaktionsgefäß überführt. Für die anschließende HIV-Bestimmung durch RT-PCR werden 40 µl Eluat verwendet.Then 100 μl of elution buffer are added and the MGP is resuspended. After 15 minutes of incubation at 80 ° C on an Eppendorf thermo mixer (13,000 RPM), 90 μl of eluate are transferred to a new reaction vessel. For the subsequent HIV determination by RT-PCR, 40 μl of eluate are used.

4.2 Semiautomatisiertes Standardprotokoll für die Probenvorbereitung4.2 Semiautomatized standard protocol for sample preparation

Die Probenvorbereitung erfolgt wie unter Punkt 4.1 beschrieben, abgesehen davon, daß das Mischen und die Temperierung auf einem Misch- und Temperiermodul erfolgte.The sample preparation is carried out as described in section 4.1, except that the mixing and the temperature control took place on a mixing and tempering module.

4.3 Semiautomatisiertes Protokoll bei Raumtemperatur4.3 Semiautomatized protocol at room temperature

Die Probenvorbereitung erfolgt im wesentlichen wie unter Punkt 4.2 beschrieben, außer daß alle Schritte bei Raumtemperatur durchgeführt werden. Die Inkubationsdauer für Lyse, Adsorption und Elution beträgt jeweils 15 min.The sample preparation is essentially as described in section 4.2, except that all steps are carried out at room temperature. The incubation period for lysis, adsorption and elution is 15 min.

Aus Abbildung 6 ist ersichtlich, daß durch die Automatisierung und die Probenvorbereitung bei Raumtemperatur (RT-Protokoll MTM) keine Beeinträchtigung der Sensitivität gegenüber dem Standardprotokoll bei manueller Probenvorbereitung (manuell) erfolgt. Sowohl bei negativen, niedrigpositiven, mittelpositiven und hochpositiven Plasmen werden reproduzierbare Ergebnisse erhalten.From Figure 6 it can be seen that the automation and sample preparation at room temperature (RT protocol MTM) does not affect the sensitivity of the standard protocol for manual sample preparation (manual). Both negative, low positive, medium positive and high positive plasmas produce reproducible results.

Claims (24)

  1. Process for the isolation of a nucleic acid analyte from a biological sample comprising the steps:
    (a) lysing the sample in a reaction vessel,
    (b) adding magnetic glass particles in the form of an alcoholic suspension,
    (c) incubating under conditions in which the analyte binds to the glass particles,
    (d) removing non-bound sample components from the reaction vessel,
    (e) incubating under conditions in which the analyte is eluted from the glass particles and
    (f) separating the eluate from the glass particles
    wherein many or all steps are carried out at essentially the same temperature.
  2. Process according to claim 1,
    characterized in that
    step (a) comprises adding a protease and a denaturing buffer.
  3. Process according to claim 2,
    characterized in that
    proteinase K is used as the protease.
  4. Process according to claim 2,
    characterized in that
    a denaturing buffer is used which contains a guanidinium salt, in particular guanidinium hydrochloride or/and guanidinium thiocyanate.
  5. Process according to one of the previous claims,
    characterized in that
    at least steps (a) to (e) are carried out at an essentially identical temperature which is in the range from room temperature to 40°C.
  6. Process according to one of the claims 1 to 5,
    characterized in that
    the alcoholic suspension has a concentration of about 5 to 20 mg/ml.
  7. Process according to claim 5 or 6,
    characterized in that
    glass particles are used whose glass phase contains SiO2, B2O3 and Na2O or SiO2 B2O3, Al2O3, CaO and K2O.
  8. Process according to one of the previous claims,
    characterized in that
    the glass particles are added in an amount that is at most 50 % more than the amount that is required to quantitatively bind the analyte present in the sample.
  9. Process according to one of the previous claims,
    characterized in that
    a continuous or intermittent mixing without adding external means is carried out at least during steps (c), (d) or/and (e).
  10. Process according to claim 9,
    characterized in that
    the mixing is achieved by rotating the reaction vessel around its longitudinal axis.
  11. Process according to claims 9 or 10,
    characterized in that
    the maximum period for carrying out steps (c) or/and (e) is 20 min in each case.
  12. Process according to one of the previous claims,
    characterized in that
    step (d) comprises adding and aspirating a wash buffer which is optionally repeated several times.
  13. Process according to claim 12,
    characterized in that
    a wash buffer is used with a content of at least 50 % (v/v) of an organic solvent that is miscible with water.
  14. Process according to one of the previous claims,
    characterized in that
    additional reagents such as enzymes are added in step (e).
  15. Process according to one of the previous claims,
    characterized in that
    a low salt buffer is used for elution in step (e).
  16. Process according to one of the claims 1 to 14,
    characterized in that
    a nucleic acid amplification master mix is added for elution in step (e).
  17. Process according to one of the claims 1 to 16,
    characterized in that
    the temperature is in the range of 18°C to 32°C.
  18. Process according to one of the previous claims,
    characterized in that
    an aftertreatment step at an elevated temperature is carried out after step (f).
  19. Process according to claim 18,
    characterized in that
    the elevated temperature is in the range from more than 40°C to 95°C.
  20. Process for the isolation of a nucleic acid analyte from a biological sample comprising the steps:
    (a) lysing the sample in a reaction vessel,
    (b) adding magnetic glass particles in the form of an alcoholic suspension,
    (c) incubating under conditions in which the analyte binds to the glass particles,
    (d) separating non-bound sample components from the glass particles,
    (e) incubating under conditions in which the analyte is eluted from the glass particles and
    (f) separating the eluate from the glass particles
    wherein many or all steps are carried out at essentially the same temperature.
  21. Process according to claim 20,
    characterized in that
    at least the steps (a) to (e) are carried out at essentially the same temperature which is in the range from room temperature to 40°C.
  22. Process according to one of the previous claims,
    characterized in that
    the process is carried out in an automated device.
  23. Process according to claim 1 to 22,
    characterized in that
    steps (a) to (e) are carried out in a single reaction vessel.
  24. Reagent kit for isolating a nucleic acid analyte, in particular to carry out the process according to one of the claims 1 to 23,
    comprising
    (a) a protease,
    (b) a sample lysing buffer,
    (c) a wash buffer,
    (d) an elution buffer and
    (e) an alcoholic suspension of magnetic glass particles.
EP98952670A 1997-10-01 1998-09-29 Method for isolating a nucleic acid Expired - Lifetime EP1019430B1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP07003878A EP1783135B1 (en) 1997-10-01 1998-09-29 Automatable method for preparing samples which can be universally applied

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE19743518A DE19743518A1 (en) 1997-10-01 1997-10-01 Automated, universally applicable sample preparation method
DE19743518 1997-10-01
PCT/EP1998/006196 WO1999016781A2 (en) 1997-10-01 1998-09-29 Automatable method for preparing samples which can be universally applied

Related Child Applications (1)

Application Number Title Priority Date Filing Date
EP07003878A Division EP1783135B1 (en) 1997-10-01 1998-09-29 Automatable method for preparing samples which can be universally applied

Publications (2)

Publication Number Publication Date
EP1019430A2 EP1019430A2 (en) 2000-07-19
EP1019430B1 true EP1019430B1 (en) 2007-07-11

Family

ID=7844362

Family Applications (2)

Application Number Title Priority Date Filing Date
EP07003878A Expired - Lifetime EP1783135B1 (en) 1997-10-01 1998-09-29 Automatable method for preparing samples which can be universally applied
EP98952670A Expired - Lifetime EP1019430B1 (en) 1997-10-01 1998-09-29 Method for isolating a nucleic acid

Family Applications Before (1)

Application Number Title Priority Date Filing Date
EP07003878A Expired - Lifetime EP1783135B1 (en) 1997-10-01 1998-09-29 Automatable method for preparing samples which can be universally applied

Country Status (13)

Country Link
US (2) US6562568B1 (en)
EP (2) EP1783135B1 (en)
JP (3) JP4048022B2 (en)
KR (1) KR20010024366A (en)
AT (1) ATE366739T1 (en)
AU (1) AU1028299A (en)
CA (1) CA2305171C (en)
DE (2) DE19743518A1 (en)
DK (1) DK1019430T3 (en)
ES (2) ES2398713T3 (en)
IL (2) IL135299A0 (en)
PT (1) PT1019430E (en)
WO (1) WO1999016781A2 (en)

Families Citing this family (72)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100463475B1 (en) * 1995-06-08 2005-06-22 로셰 디아그노스틱스 게엠베하 Magnetic Pigment
CA2318306A1 (en) * 1997-12-06 1999-06-17 Dna Research Instruments Limited Isolation of nucleic acids
AU2862100A (en) * 1999-01-27 2000-08-18 Folim G. Halaka Materials and methods for the purification of polyelectrolytes
DE19937607A1 (en) * 1999-08-09 2001-02-15 Bilatec Ges Zur Entwicklung Bi Laboratory robot, useful for automated isolation of nucleic acid, includes orientation device to ensure proper alignment between robotic arm and unit being transported
JP2003514383A (en) * 1999-11-17 2003-04-15 ロシュ ダイアグノスティクス ゲゼルシャフト ミット ベシュレンクテル ハフツング Magnetic glass particles, their production method and their use
JP2004515219A (en) * 2000-06-15 2004-05-27 ボード・オブ・リージェンツ，ザ・ユニヴァーシティ・オヴ・テキサス・システム Tunable catalytically active nucleic acids
US7494817B2 (en) * 2000-09-06 2009-02-24 Transnet Yx, Inc. Methods for genotype screening using magnetic particles
DE10127068A1 (en) * 2001-05-23 2002-11-28 Bio Medical Apherese Systeme G Apparatus for the separation of leukocytes, from blood, has a number of stations to separate marked components with magnetic particles without any adverse effects on the blood
DE10147439B4 (en) * 2001-09-26 2014-01-30 Qiagen Gmbh Method for isolating DNA from biological samples
DE60316660T3 (en) 2002-01-08 2016-01-28 Roche Diagnostics Gmbh USE OF A SILICA MATERIAL IN AMPLIFICATION
AU2003202026A1 (en) 2002-01-16 2003-09-02 Dynal Biotech Asa Method for isolating nucleic acids and protein from a single sample
US20040234898A1 (en) * 2002-02-06 2004-11-25 Batishko Charles R. Magnetic flowcell systems and methods
ATE516366T1 (en) 2002-07-25 2011-07-15 Archemix Corp REGULATED APTAMER THERAPEUTICS
AU2003236461B2 (en) 2002-08-29 2009-05-28 Epigenomics Ag Improved method for bisulfite treatment
EP1590362B8 (en) 2003-01-29 2015-06-03 Epigenomics AG Improved method for bisulfite treatment
US7601491B2 (en) 2003-02-06 2009-10-13 Becton, Dickinson And Company Pretreatment method for extraction of nucleic acid from biological samples and kits therefor
JP3644641B2 (en) * 2003-03-07 2005-05-11 株式会社日立ハイテクノロジーズ Nucleic acid recovery method
JP4871722B2 (en) † 2003-07-25 2012-02-08 アプライド バイオシステムズ リミテッド ライアビリティー カンパニー Methods and compositions for preparing RNA from fixed samples
US20050042660A1 (en) * 2003-07-31 2005-02-24 Hall Gerald Edward Devices and methods for isolating RNA
GB0319671D0 (en) * 2003-08-21 2003-09-24 Secr Defence Apparatus for processing a fluid sample
CA2482097C (en) 2003-10-13 2012-02-21 F. Hoffmann-La Roche Ag Methods for isolating nucleic acids
WO2005054502A1 (en) 2003-12-02 2005-06-16 Roche Diagnostics Gmbh Improved method for bisulfite treatment
JP2007516729A (en) * 2003-12-30 2007-06-28 シグマ−アルドリッチ・カンパニー Rapid preparation of nucleic acids by enzymatic digestion
CA2791798C (en) 2004-02-10 2016-08-16 F. Hoffmann-La Roche Ag New primers and probes for the detection of parvovirus b19
US9267167B2 (en) 2004-06-28 2016-02-23 Becton, Dickinson And Company Dissolvable films and methods including the same
US20060099605A1 (en) * 2004-11-11 2006-05-11 Hall Gerald E Jr Devices and methods for isolating RNA
EP1632578A1 (en) 2004-09-03 2006-03-08 Roche Diagnostics GmbH DNA decontamination method
EP1690938A1 (en) * 2005-02-11 2006-08-16 Qiagen GmbH Method for isolating nucleic acids, wherein the nucleic acids are immobilised on a matrix at elevated temperatures
US20060270843A1 (en) * 2005-05-26 2006-11-30 Hall Gerald E Jr Methods for isolation of nucleic acids
US20060269929A1 (en) * 2005-05-26 2006-11-30 Hall Gerald E Jr Methods and kits for DNA purification on polymeric membranes at low ionic strength
JP4733456B2 (en) * 2005-07-26 2011-07-27 シスメックス株式会社 External quality control method for preprocessing, external quality control method for nucleic acid detection processing, external quality control method for calibration curve creation processing, external quality control computer for preprocessing, external quality control computer for nucleic acid detection processing, external of calibration curve creation processing Quality control computer, pretreatment device, and nucleic acid detection device
GB0525231D0 (en) * 2005-12-12 2006-01-18 Genpoint As Method
US7981606B2 (en) 2005-12-21 2011-07-19 Roche Molecular Systems, Inc. Control for nucleic acid testing
FR2895993A1 (en) * 2006-01-10 2007-07-13 Immunid Sarl COMPOSITION FOR STABILIZING BIOLOGICAL SAMPLES AND METHOD OF STABILIZATION
US20090155811A1 (en) * 2006-01-27 2009-06-18 Oxonica, Inc. Lateral Flow Immunoassay With Encapsulated Detection Modality
EP3604553A3 (en) 2006-11-03 2020-04-29 Siemens Healthcare Diagnostics Inc. Random access system for polymerase chain reaction testing
JP5354894B2 (en) 2006-12-11 2013-11-27 エフ．ホフマン−ラ ロシュ アーゲー Nucleic acid isolation using polidocanols and derivatives
DE602007010986D1 (en) 2006-12-13 2011-01-13 Roche Diagnostics Gmbh USE OF ACETALES FOR THE ISOLATION OF NUCLEIC ACIDS
ES2394815T3 (en) 2006-12-13 2013-02-05 F. Hoffmann-La Roche Ag Use of TDE for nucleic acid isolation
US8535888B2 (en) 2006-12-29 2013-09-17 Mayo Foundation For Medical Education And Research Compositions and methods for detecting methicillin-resistant S. aureus
US7985375B2 (en) * 2007-04-06 2011-07-26 Qiagen Gaithersburg, Inc. Sample preparation system and method for processing clinical specimens
US8703492B2 (en) 2007-04-06 2014-04-22 Qiagen Gaithersburg, Inc. Open platform hybrid manual-automated sample processing system
DE102008010693A1 (en) * 2008-02-22 2009-08-27 Qiagen Gmbh New matrix materials, processes for their preparation and their use in methods of isolating biomolecules
JP5572939B2 (en) * 2008-10-27 2014-08-20 株式会社島津製作所 DNA sequencing method
US8911663B2 (en) 2009-03-05 2014-12-16 Quebec Metal Powders, Ltd. Insulated iron-base powder for soft magnetic applications
TWI407994B (en) 2009-10-22 2013-09-11 Ind Tech Res Inst Method, agent, and kit for isolating nucleic acids
WO2011063139A1 (en) 2009-11-18 2011-05-26 Qiagen Laboratory central control unit method and system
US8609340B2 (en) 2010-07-29 2013-12-17 Roche Molecular Systems, Inc. Control nucleic acids for multiple parameters
JP6348202B2 (en) * 2010-07-29 2018-06-27 エフ．ホフマン−ラ ロシュ アーゲーＦ． Ｈｏｆｆｍａｎｎ−Ｌａ Ｒｏｃｈｅ Ａｋｔｉｅｎｇｅｓｅｌｌｓｃｈａｆｔ General sample preparation
CA2802563C (en) 2010-07-29 2018-08-28 F. Hoffmann-La Roche Ag Preparation of nucleic acids from different types of biological fluid samples
US9175332B2 (en) 2010-07-29 2015-11-03 Roche Molecular Systems, Inc. Generic PCR
US9725754B2 (en) 2010-07-29 2017-08-08 Sean F. Boyle Generic sample preparation
EP2707381B1 (en) * 2011-05-11 2019-08-14 Exosome Diagnostics, Inc. Nucleic acid extraction from heterogeneous biological materials
KR102010106B1 (en) * 2011-11-09 2019-08-12 더 리전트 오브 더 유니버시티 오브 캘리포니아 Superparamagnetic colloids with enhanced charge stability for high quality magnetically tunable photonic structures
EP2664914A1 (en) * 2012-05-16 2013-11-20 Koninklijke Philips N.V. Magnetically assisted processing of a medium
US9399986B2 (en) 2012-07-31 2016-07-26 General Electric Company Devices and systems for isolating biomolecules and associated methods thereof
US20140322706A1 (en) 2012-10-24 2014-10-30 Jon Faiz Kayyem Integrated multipelx target analysis
EP2965817B1 (en) 2012-10-24 2017-09-27 Genmark Diagnostics Inc. Integrated multiplex target analysis
US9222623B2 (en) 2013-03-15 2015-12-29 Genmark Diagnostics, Inc. Devices and methods for manipulating deformable fluid vessels
US9498778B2 (en) 2014-11-11 2016-11-22 Genmark Diagnostics, Inc. Instrument for processing cartridge for performing assays in a closed sample preparation and reaction system
USD881409S1 (en) 2013-10-24 2020-04-14 Genmark Diagnostics, Inc. Biochip cartridge
JP5928435B2 (en) 2013-11-01 2016-06-01 株式会社安川電機 Robot system, inspection method, and inspection object production method
CN105255855A (en) * 2014-07-10 2016-01-20 上海艾迪康医学检验所有限公司 Method for preserving nucleic acid on solid surface, and preservation solution thereof
US10005080B2 (en) 2014-11-11 2018-06-26 Genmark Diagnostics, Inc. Instrument and cartridge for performing assays in a closed sample preparation and reaction system employing electrowetting fluid manipulation
US9598722B2 (en) 2014-11-11 2017-03-21 Genmark Diagnostics, Inc. Cartridge for performing assays in a closed sample preparation and reaction system
WO2016077294A1 (en) 2014-11-14 2016-05-19 Corning Incorporated Methods and kits for post-ivt rna purification
EP3434372B1 (en) * 2014-11-19 2020-02-26 F. Hoffmann-La Roche AG Particle mixing
US10001501B2 (en) 2015-12-31 2018-06-19 Universal Diagnostics, S. L. Systems and methods for automated, customizable sample preparation for detection of metabolites and lipids
CN108474024B (en) * 2016-01-05 2022-08-02 豪夫迈·罗氏有限公司 Continuous capture of nucleic acids by magnetic glass particles
ES2951666T3 (en) * 2016-09-12 2023-10-24 Hoffmann La Roche Procedures and compositions for purifying double-stranded nucleic acids
WO2019014486A1 (en) 2017-07-12 2019-01-17 Exosome Diagnostics, Inc. Methods for isolating and enriching populations of biofluid- derived extracellular vesicles, and methods of use thereof
CN114381368B (en) * 2021-12-31 2023-10-27 北京理工大学 System and method for automatically separating biological analytes by utilizing gas-liquid interface

Family Cites Families (97)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2913419A (en) * 1956-04-18 1959-11-17 Du Pont Chemical process and composition
US2885366A (en) * 1956-06-28 1959-05-05 Du Pont Product comprising a skin of dense, hydrated amorphous silica bound upon a core of another solid material and process of making same
DE2313331C2 (en) * 1973-03-17 1986-11-13 Merck Patent Gmbh, 6100 Darmstadt Mica flake pigments containing iron oxide
US3945862A (en) * 1973-06-26 1976-03-23 Merck & Co., Inc. Coated ferrous substrates comprising an amorphous magnesia-silica complex
US4133169A (en) * 1974-08-30 1979-01-09 Ebauches S.A. Electronic circuit for a quartz crystal watch
DE2625106C2 (en) * 1976-06-04 1982-03-11 Bayer Ag, 5090 Leverkusen Iron oxide black pigments with improved oxidation resistance and process for their preparation
US4124385A (en) * 1976-12-02 1978-11-07 Xerox Corporation Magnetic glass carrier materials
US4126437A (en) * 1976-12-02 1978-11-21 Xerox Corporation Magnetic glass carrier materials
US4124735A (en) * 1976-12-02 1978-11-07 Xerox Corporation Magnetic glass carrier materials
US4395271A (en) * 1979-04-13 1983-07-26 Corning Glass Works Method for making porous magnetic glass and crystal-containing structures
US4297337A (en) * 1979-04-13 1981-10-27 Corning Glass Works Solid-phase immunoassays using magnetic glass
JPS5676510A (en) * 1979-11-28 1981-06-24 Tdk Corp Manufacture of magnetic recording material
JPS56105337A (en) * 1980-01-28 1981-08-21 Tdk Corp Magnetic recording medium and its production
US4280918A (en) * 1980-03-10 1981-07-28 International Business Machines Corporation Magnetic particle dispersions
US4360441A (en) * 1981-06-25 1982-11-23 Corning Glass Works Glass-encapsulated magnetic materials and methods for making them
US4454234A (en) * 1981-12-30 1984-06-12 Czerlinski George H Coated magnetizable microparticles, reversible suspensions thereof, and processes relating thereto
US4448884A (en) * 1982-03-03 1984-05-15 Kms Fusion, Inc. Glass-surface microcarrier for growth of cell cultures
US4477492A (en) * 1983-04-22 1984-10-16 E. I. Du Pont De Nemours And Company Process for preparing superficially porous supports for chromatography and catalysts
US4628037A (en) * 1983-05-12 1986-12-09 Advanced Magnetics, Inc. Binding assays employing magnetic particles
US4672040A (en) * 1983-05-12 1987-06-09 Advanced Magnetics, Inc. Magnetic particles for use in separations
US4695392A (en) * 1983-05-12 1987-09-22 Advanced Magnetics Inc. Magnetic particles for use in separations
US4698302A (en) * 1983-05-12 1987-10-06 Advanced Magnetics, Inc. Enzymatic reactions using magnetic particles
US4695393A (en) * 1983-05-12 1987-09-22 Advanced Magnetics Inc. Magnetic particles for use in separations
US4554088A (en) * 1983-05-12 1985-11-19 Advanced Magnetics Inc. Magnetic particles for use in separations
GB8401636D0 (en) * 1984-01-21 1984-02-22 British Petroleum Co Plc Coating process
US5236623A (en) * 1984-07-11 1993-08-17 Rhone-Poulenc Chimie Process for the production of a silica colloid
US4824712A (en) * 1984-07-16 1989-04-25 Ppg Industries, Inc. Treatment of glass to reduce venting during thermal treatment and a glass article made thereby
US4748121A (en) * 1984-11-30 1988-05-31 Ppg Industries, Inc. Porous glass fibers with immobilized biochemically active material
US4683195A (en) * 1986-01-30 1987-07-28 Cetus Corporation Process for amplifying, detecting, and/or-cloning nucleic acid sequences
US4683202A (en) * 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
US5512332A (en) * 1985-10-04 1996-04-30 Immunivest Corporation Process of making resuspendable coated magnetic particles
US5597531A (en) * 1985-10-04 1997-01-28 Immunivest Corporation Resuspendable coated magnetic particles and stable magnetic particle suspensions
US5076950A (en) * 1985-12-20 1991-12-31 Syntex (U.S.A.) Inc. Magnetic composition for particle separation
US5206568A (en) * 1986-03-26 1993-04-27 Beckman Instruments, Inc. Coordinated control of stepper motors
US4751211A (en) * 1986-08-07 1988-06-14 Aluminum Company Of America Composite adsorbent for removing acids from organophosphate functional fluids
JPS63109105A (en) * 1986-10-25 1988-05-13 Chisso Corp Production of fine ferromagnetic metal particle
DE3639949A1 (en) * 1986-11-22 1988-06-09 Diagen Inst Molekularbio METHOD FOR SEPARATING LONG CHAIN NUCLEIC ACIDS
US4699171A (en) * 1986-12-19 1987-10-13 Sundstrand Corporation Multiple port relief valve
US4767670A (en) * 1987-01-21 1988-08-30 E. I. Du Pont De Nemours And Company Chromatographic supports for separation of oligonucleotides
NO162946C (en) * 1987-08-21 1990-03-14 Otto Soerensen DEVICE FOR MAGNETIC SEPARATION OF CELLS.
CH672445A5 (en) 1987-02-27 1989-11-30 Hratch Boyadjian
JP2862547B2 (en) * 1987-03-02 1999-03-03 ジエン―プローブ・インコーポレイテツド Polycationic support for nucleic acid purification, separation and hybridization
DE68920778T2 (en) * 1988-05-24 1995-05-18 Anagen Uk Ltd Magnetically attractable particles and manufacturing processes.
US5312485A (en) * 1988-08-05 1994-05-17 J. M. Huber Corporation Precipitated encapsulated paper pigments and methods
US5041390A (en) * 1988-08-11 1991-08-20 Skov Per S Method and means for allergy diagnosis
US5075430A (en) * 1988-12-12 1991-12-24 Bio-Rad Laboratories, Inc. Process for the purification of DNA on diatomaceous earth
US5234809A (en) 1989-03-23 1993-08-10 Akzo N.V. Process for isolating nucleic acid
US5352645A (en) * 1989-04-14 1994-10-04 E. I. Du Pont De Nemours And Company Silica microspheres, method of improving attrition resistance and use
US5055194A (en) * 1989-07-28 1991-10-08 University Of Pennsylvania Support for high performance liquid chromatography in a magnetically stabilized fluidized bed
US5279936A (en) * 1989-12-22 1994-01-18 Syntex (U.S.A.) Inc. Method of separation employing magnetic particles and second medium
US5523231A (en) * 1990-02-13 1996-06-04 Amersham International Plc Method to isolate macromolecules using magnetically attractable beads which do not specifically bind the macromolecules
US5236783A (en) * 1990-02-21 1993-08-17 Toda Kogyo Corp. Superparamagnetic fine particles of iron oxide and magnetic recording media containing said particles
US5316699A (en) * 1990-03-28 1994-05-31 The United States Of America As Repesented By The Secretary Of Commerce Process for the controlled preparation of a composite of ultrafine magnetic particles homogeneously dispersed in a dielectric matrix
EP0478753B1 (en) * 1990-04-06 1997-07-02 The Perkin-Elmer Corporation Automated molecular biology laboratory
US5763173A (en) * 1990-06-11 1998-06-09 Nexstar Pharmaceuticals, Inc. Nucleic acid ligand inhibitors to DNA polymerases
US5210015A (en) * 1990-08-06 1993-05-11 Hoffman-La Roche Inc. Homogeneous assay system using the nuclease activity of a nucleic acid polymerase
US5217804A (en) * 1990-11-06 1993-06-08 Eastman Kodak Company Magnetic particles
US5155018A (en) * 1991-07-10 1992-10-13 Hahnemann University Process and kit for isolating and purifying RNA from biological sources
US5395498A (en) * 1991-11-06 1995-03-07 Gombinsky; Moshe Method for separating biological macromolecules and means therfor
US5734020A (en) * 1991-11-20 1998-03-31 Cpg, Inc. Production and use of magnetic porous inorganic materials
US5610274A (en) * 1991-11-20 1997-03-11 Cpg, Inc. Production and use of magnetic porous inorganic materials
US5346994A (en) * 1992-01-28 1994-09-13 Piotr Chomczynski Shelf-stable product and process for isolating RNA, DNA and proteins
EP0566790B1 (en) * 1992-04-23 1996-08-07 Toda Kogyo Corp. Magnetic powder and magnetic toner
TW221381B (en) * 1992-04-28 1994-03-01 Du Pont
IT1256064B (en) * 1992-07-28 1995-11-27 Donegani Guido Ist METHOD FOR THE PREPARATION OF POROUS GELS CONTAINING BORON
CA2107524C (en) * 1992-10-06 1999-01-19 Hiromitsu Misawa Iron oxide particles and process for producing the same
US5578238A (en) * 1992-10-30 1996-11-26 Lord Corporation Magnetorheological materials utilizing surface-modified particles
AU669398B2 (en) * 1992-11-13 1996-06-06 Becton Dickinson & Company Boron silicates aluminum silicates phosphosilicates and purification of DNA
US5648170A (en) * 1993-04-27 1997-07-15 Toda Kogyo Corporation Coated granular magnetite particles and process for producing the same
EP0723549B1 (en) * 1993-08-30 2003-12-17 Promega Corporation Nucleic acid purification compositions and methods
US5503816A (en) * 1993-09-27 1996-04-02 Becton Dickinson And Company Silicate compounds for DNA purification
US5438127A (en) * 1993-09-27 1995-08-01 Becton Dickinson And Company DNA purification by solid phase extraction using a PCl3 modified glass fiber membrane
US5599627A (en) * 1993-10-08 1997-02-04 Toda Kogyo Corporation Magnetic particles comprising magnetite core and process for producing the same
US5538848A (en) * 1994-11-16 1996-07-23 Applied Biosystems Division, Perkin-Elmer Corp. Method for detecting nucleic acid amplification using self-quenching fluorescence probe
US5990301A (en) * 1994-02-07 1999-11-23 Qiagen Gmbh Process for the separation and purification of nucleic acids from biological sources
AU691574B2 (en) * 1994-02-07 1998-05-21 Qiagen Gmbh Process for producing endotoxin-free or endotoxin-poor nucleic acids and/or oligonucleotides for gene therapy
GB9411572D0 (en) * 1994-06-09 1994-08-03 Amersham Int Plc Magnetic bead precipitation method
ATE191086T1 (en) * 1994-07-27 2000-04-15 Pilgrimm Herbert SUPERPARAMAGNETIC PARTICLES, METHOD FOR THE PRODUCTION AND USE THEREOF
US5705628A (en) * 1994-09-20 1998-01-06 Whitehead Institute For Biomedical Research DNA purification and isolation using magnetic particles
US5660984A (en) * 1994-12-09 1997-08-26 Davis; Thomas E. DNA isolating apparatus comprising a non-porous DNA binding, anion exchange resin and methods of use thereof
FR2732116B1 (en) * 1995-03-21 1997-05-09 Bio Merieux METHOD AND DEVICE FOR THE QUALITATIVE AND / OR QUANTITATIVE DETERMINATION OF AN ANALYTE, IN PARTICULAR OF A BACTERIA, IN A SAMPLE, BY MAGNETIC WAY
US5683875A (en) * 1995-05-04 1997-11-04 Hewlett-Packard Company Method for detecting a target nucleic acid analyte in a sample
IN188702B (en) * 1995-06-01 2002-10-26 Degussa
DE19520964A1 (en) 1995-06-08 1996-12-12 Inst Neue Mat Gemein Gmbh Coated inorganic pigments, process for their preparation and their use
DE19520398B4 (en) * 1995-06-08 2009-04-16 Roche Diagnostics Gmbh Magnetic pigment
JP2965131B2 (en) 1995-07-07 1999-10-18 東洋紡績株式会社 Magnetic carrier for nucleic acid binding and nucleic acid isolation method using the same
CA2231861A1 (en) 1995-09-13 1997-03-20 Takanori Oka Method for concentrating mutant nucleic acid and nucleic acid-concentrating assay kit for said concentration method
US5783686A (en) 1995-09-15 1998-07-21 Beckman Instruments, Inc. Method for purifying nucleic acids from heterogenous mixtures
US5904848A (en) * 1996-02-21 1999-05-18 Cpg, Inc. Controlled pore glass-synthetic resin membrane
US5972721A (en) * 1996-03-14 1999-10-26 The United States Of America As Represented By The Secretary Of The Air Force Immunomagnetic assay system for clinical diagnosis and other purposes
DE19622885A1 (en) * 1996-06-07 1997-12-11 Boehringer Mannheim Gmbh Reagent preparation containing magnetic particles in the form of a tablet
AU6021798A (en) 1997-01-21 1998-08-07 W.R. Grace & Co.-Conn. Silica adsorbent on magnetic substrate
US6027945A (en) 1997-01-21 2000-02-22 Promega Corporation Methods of isolating biological target materials using silica magnetic particles
US5990479A (en) * 1997-11-25 1999-11-23 Regents Of The University Of California Organo Luminescent semiconductor nanocrystal probes for biological applications and process for making and using such probes
US6242235B1 (en) 1998-06-24 2001-06-05 Promega Corp. Polymerase stabilization by polyethoxylated amine surfactants
DK1144620T3 (en) * 1998-11-30 2004-01-05 Roche Diagnostics Gmbh Magnetic particles for the purification of nucleic acids
US7267964B2 (en) * 2000-03-31 2007-09-11 Merial Limited DNA molecules encoding ligand gated ion channels from Dermacentor variabilis

Also Published As

Publication number Publication date
IL135299A0 (en) 2001-05-20
PT1019430E (en) 2007-10-16
JP4659013B2 (en) 2011-03-30
JP2008134239A (en) 2008-06-12
WO1999016781A2 (en) 1999-04-08
DE19743518A1 (en) 1999-04-15
DE59814056D1 (en) 2007-08-23
WO1999016781A3 (en) 1999-09-16
ATE366739T1 (en) 2007-08-15
IL135299A (en) 2010-11-30
DK1019430T3 (en) 2007-11-12
CA2305171C (en) 2008-06-17
ES2290996T3 (en) 2008-02-16
AU1028299A (en) 1999-04-23
JP4048022B2 (en) 2008-02-13
JP2001518284A (en) 2001-10-16
US6562568B1 (en) 2003-05-13
EP1783135A1 (en) 2007-05-09
CA2305171A1 (en) 1999-04-08
EP1019430A2 (en) 2000-07-19
JP2007175060A (en) 2007-07-12
ES2398713T3 (en) 2013-03-21
KR20010024366A (en) 2001-03-26
EP1783135B1 (en) 2012-11-07
US20030199078A1 (en) 2003-10-23

Similar Documents

Publication Publication Date Title
EP1019430B1 (en) Method for isolating a nucleic acid
EP2998398B1 (en) Automated system for purifying nucleic acids, particularly from fixed tissue
EP1577389B1 (en) Magnetic pigment
EP2288701B1 (en) Method for filtering nucleic acids, in particular from fixed tissue
EP1851313B1 (en) Method for isolating nucleic acids, wherein the nucleic acids are immobilised on a matrix at elevated temperatures
DE69533317T2 (en) ISOLATION OF NUCLEIC ACIDS
DE60316660T2 (en) USE OF A SILICA MATERIAL IN AMPLIFICATION
EP1135479B1 (en) Formulations and methods for isolating nucleic acids from any complex starting material and subsequent complex genetic analysis
DE69802191T3 (en) Solid phase nucleic acid isolation
DE69734263T2 (en) Process for isolating ribonucleic acids.
DE19858447A1 (en) Isolating and purifying nucleic acid
JPH11196869A (en) Isolation of liponucleic acid
DE10103652A1 (en) Magnetic polyvinyl alcohol carrier, useful for isolating and purifying nucleic acid, is modified with germanium compound for improved reversible binding
CZ20001132A3 (en) Automated, universally usable process for preparing samples
MXPA00003007A (en) Automatable method for preparing samples which can be universally applied

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20000502

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LI LU NL PT SE

17Q First examination report despatched

Effective date: 20020626

17Q First examination report despatched

Effective date: 20020626

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

RTI1 Title (correction)

Free format text: METHOD FOR ISOLATING A NUCLEIC ACID

GRAS Grant fee paid

Free format text: ORIGINAL CODE: EPIDOSNIGR3

GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

AK Designated contracting states

Kind code of ref document: B1

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LI LU NL PT SE

REG Reference to a national code

Ref country code: GB

Ref legal event code: FG4D

Free format text: NOT ENGLISH

REG Reference to a national code

Ref country code: CH

Ref legal event code: EP

REF Corresponds to:

Ref document number: 59814056

Country of ref document: DE

Date of ref document: 20070823

Kind code of ref document: P

REG Reference to a national code

Ref country code: IE

Ref legal event code: FG4D

Free format text: LANGUAGE OF EP DOCUMENT: GERMAN

REG Reference to a national code

Ref country code: CH

Ref legal event code: NV

Representative=s name: F. HOFFMANN-LA ROCHE AG

REG Reference to a national code

Ref country code: PT

Ref legal event code: SC4A

Free format text: AVAILABILITY OF NATIONAL TRANSLATION

Effective date: 20071002

REG Reference to a national code

Ref country code: GR

Ref legal event code: EP

Ref document number: 20070402933

Country of ref document: GR

GBT Gb: translation of ep patent filed (gb section 77(6)(a)/1977)

Effective date: 20071003

REG Reference to a national code

Ref country code: SE

Ref legal event code: TRGR

REG Reference to a national code

Ref country code: DK

Ref legal event code: T3

ET Fr: translation filed
REG Reference to a national code

Ref country code: ES

Ref legal event code: FG2A

Ref document number: 2290996

Country of ref document: ES

Kind code of ref document: T3

PLBE No opposition filed within time limit

Free format text: ORIGINAL CODE: 0009261

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT

26N No opposition filed

Effective date: 20080414

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: DK

Payment date: 20110825

Year of fee payment: 14

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: IE

Payment date: 20120829

Year of fee payment: 15

Ref country code: LU

Payment date: 20120926

Year of fee payment: 15

Ref country code: SE

Payment date: 20120913

Year of fee payment: 15

Ref country code: FI

Payment date: 20120907

Year of fee payment: 15

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: GR

Payment date: 20120830

Year of fee payment: 15

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: BE

Payment date: 20121001

Year of fee payment: 15

Ref country code: NL

Payment date: 20120913

Year of fee payment: 15

Ref country code: PT

Payment date: 20120329

Year of fee payment: 15

BERE Be: lapsed

Owner name: ROCHE DIAGNOSTICS G.M.B.H.

Effective date: 20130930

REG Reference to a national code

Ref country code: PT

Ref legal event code: MM4A

Free format text: LAPSE DUE TO NON-PAYMENT OF FEES

Effective date: 20140331

REG Reference to a national code

Ref country code: NL

Ref legal event code: V1

Effective date: 20140401

REG Reference to a national code

Ref country code: SE

Ref legal event code: EUG

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: FI

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20130929

Ref country code: SE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20130930

REG Reference to a national code

Ref country code: DK

Ref legal event code: EBP

Effective date: 20130930

REG Reference to a national code

Ref country code: GR

Ref legal event code: ML

Ref document number: 20070402933

Country of ref document: GR

Effective date: 20140403

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: PT

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20140331

REG Reference to a national code

Ref country code: IE

Ref legal event code: MM4A

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: BE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20130930

Ref country code: IE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20130929

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: GR

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20140403

Ref country code: NL

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20140401

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: DK

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20130930

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LU

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20130929

REG Reference to a national code

Ref country code: FR

Ref legal event code: PLFP

Year of fee payment: 19

REG Reference to a national code

Ref country code: FR

Ref legal event code: PLFP

Year of fee payment: 20

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: IT

Payment date: 20170913

Year of fee payment: 20

Ref country code: GB

Payment date: 20170829

Year of fee payment: 20

Ref country code: CH

Payment date: 20170725

Year of fee payment: 20

Ref country code: FR

Payment date: 20170823

Year of fee payment: 20

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: AT

Payment date: 20170828

Year of fee payment: 20

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: DE

Payment date: 20170928

Year of fee payment: 20

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: ES

Payment date: 20171004

Year of fee payment: 20

REG Reference to a national code

Ref country code: DE

Ref legal event code: R071

Ref document number: 59814056

Country of ref document: DE

REG Reference to a national code

Ref country code: CH

Ref legal event code: PL

REG Reference to a national code

Ref country code: GB

Ref legal event code: PE20

Expiry date: 20180928

REG Reference to a national code

Ref country code: AT

Ref legal event code: MK07

Ref document number: 366739

Country of ref document: AT

Kind code of ref document: T

Effective date: 20180929

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: GB

Free format text: LAPSE BECAUSE OF EXPIRATION OF PROTECTION

Effective date: 20180928

REG Reference to a national code

Ref country code: ES

Ref legal event code: FD2A

Effective date: 20200904

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: ES

Free format text: LAPSE BECAUSE OF EXPIRATION OF PROTECTION

Effective date: 20180930